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Bacterial cell lysis buffer used in proteomics procedures

Bacterial cell lysis buffer used in proteomics procedures


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What kinds of detergent-free bacterial lysis buffers exist? The proteins we're extracting will be later analyzed by LC-MS/MS, and we're looking for a lysis buffer that won't interfere with this downstream analysis.


I'm a HUGE fan of FASP (filter-aided sample preparation) which is an in-solution preparation/digestion. It is very fast, allows you to not worry about a protein precipitation step, and gets rid of all MS incompatible substances with the wash steps (so doesn't matter what lysis buffer you use). It requires very few, inexpensive materials (a pack of 100 centrifuge filter units is less than 250 USD from VWR) and has great recovery. This is the link to the Nature Methods paper that the linked protocol is based on (the linked protocol is an updated, faster version of the Nature Methods one).


There are a few techniques available for this.

Some recommend a urea-based technique, others recommend a chloroform. Do you have more information about your specific needs?

A Chloroform-Assisted Protein Isolation Method Followed by Capillary NanoLC-MS Identify Estrogen-Regulated Proteins from MCF7 Cells


why not use a french press? put a few thousand PSI on the sample and the cells just pop open. The lysis buffer contains no detergents:

Lysis buffer: 50 mM Tris-HCl pH 7.5 50-200 mM NaCl* 1 mM MgCl2 5 mM DTT 1 mM PMSF

the salt concentration is variable and you could probably do without the DTT too if thats a problem…


As pstew mentioned, SDS based lysis followed by FASP is a very good way. However, FASP can lead to sample losses. I would use it only if you have large culture of bacterial cells.

If you want totally detergent free method then, lyse cells in 8 M guanidinium hydrochloride buffered to pH 7.5-8.2 using 50 mM Tris buffer or 50 mM freshly prepared ammonium bicarbonate. If your cell pellet is approximately 20 uL then add 60 uL 8 M GuHCL buffered solution so that final conc of GuHCL is ~ 6M, heat to 65 degrees C and shake for 30 minutes or so. Spin down to remove any insoluble debris. Measure protein concentraion, if you want to, using BCA assay. Then, reduce with 10 mM DTT for 30 minutes at 56 C, alkylate with 25 mM iodoacetamide at room temperature and in dark (for this step it is crucial that the pH of your sample is around 8.0 otherwise iodoacetamide will cause side reactions). Dilute at least 8 times and digest overnight with Trypsin.


Freeze/Thaw procedure in cell lysis - How is this done. (Jul/10/2006 )

Hello,
I need to do do a western blot in MCF7 cells, and I am confused about how to do the cell protein extracts. I will use to lysate RIPA buffer and the freeze/thaw procedure, but I dont know the order in which i have to do this. I have this protocol, im not sure if its right. Please help me.
1. wash cells in cold PBS and remove the PBS
2. add 10ml of iced cold PBS and scrape the cells , pellet the cells and resuspend in RIPA
3. aliquot and incubate on ice for 30min
4. sonicate <------ I can not sonicate, I need to freeze/thaw, but I dont know where in this steps should it go, and how to do it (should I freeze/thaw in RIPA buffer? or PBS?? )
5.centrifuge , remove supernatant and store @ -80

Please, could anybody tell me how to do this??

Freeze and thaw 3X in any buffer is sufficient to break cells.

What is the compostion of RIPA? RIPA may be better choice if contains protease inhibitors.

Unless your particular protocol requires this as a treatment step, you dont need to keep cell suspension in RIPA for 30 min on ice. you can go directly to F-T 3X.

You can also add lysozyme and DNase to your lysis buffer, both of which speed the lysis /genomic DNA shearing steps, sos yuo get to move on to the fun bits, liek purifying the protein.

Yes, you add protease inhibitors to RIPA:
* 150 mM NaCl
* 50 mM Tris, pH 7.4
* 1 % NP-40
* 0.25 % Sodium deoxycholate
* EDTA 1 mM
when I look for RIPA recipe they are all different, some use 50mM HEPES instead of Tris, is there a difference in this?

Thanks a lot, your suggestion clarified my mind.

Freeze and thaw 3X in any buffer is sufficient to break cells.

What is the compostion of RIPA? RIPA may be better choice if contains protease inhibitors.

Unless your particular protocol requires this as a treatment step, you dont need to keep cell suspension in RIPA for 30 min on ice. you can go directly to F-T 3X.

Your mixture already has very strong membrane lytic activity, it should be sufficient to lyse cells in 5-10 min on ice and F-T is not needed. PBS does not contain detergents, such as NP-40 and SDS, so FT 3X will be needed.

Buffer tris and hepes makes little difference, other than that tris has a weak amine and may react with amine-reactive agents, such as glutaldehyde. You dont have to worry about it for your cell lysis experiment.

I suggest that you add protease cooktail to reduce protein degradation.

If you are studying cytosolic proteins, this will be enough for sample prep.

If you don't have a sonicator, you can also pass your extracts through the needle of a siringe. You should use the 1ml siringe with the very small needle (like the one people use for insulin or drugs)

as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity

This is great!! thank you very much for the suggestions.

I feel so happy I count with the support and advise of all of you!!

Your mixture already has very strong membrane lytic activity, it should be sufficient to lyse cells in 5-10 min on ice and F-T is not needed. PBS does not contain detergents, such as NP-40 and SDS, so FT 3X will be needed.

Buffer tris and hepes makes little difference, other than that tris has a weak amine and may react with amine-reactive agents, such as glutaldehyde. You dont have to worry about it for your cell lysis experiment.

I suggest that you add protease cooktail to reduce protein degradation.

If you are studying cytosolic proteins, this will be enough for sample prep.

I am making RIPA from scratch, I won't have manufacturer instructions, but I dont think I will be adding lysozyme and DNase.

as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity

I am making RIPA from scratch, I won't have manufacturer instructions, but I dont think I will be adding lysozyme and DNase.

as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity

I tried adding DNase though manufacturer said it wouldn't work and it made no difference on the gl so just leave it be.


Related Technical Articles

Numerous methods are available for disrupting cells and preparing their contents for analysis. In general, gentle methods are employed when the sample consists of easily lysed cultured cells or blood cells, whereas more vigorous methods are employed for the disruption of more robust bacterial or plant cells, or mammalian cells embedded in connective tissue.

    Detergent-based lysis: Detergent-based lysis: Detergent lysis is a gentle method that can be used for mammalian cells, bacterial cells, yeasts, and plants. Cell suspensions are gently centrifuged and resuspended in lysis solution containing detergents which act to disrupt the cell membrane. The membranes are solubilized, lysing cells and liberating their contents. Detergents may need to be removed downstream if they interfere with analysis or production. Freeze-thaw lysis: This method is applicable to suspensions of mammalian or bacterial cells. The cell suspension is rapidly frozen using liquid nitrogen. The sample is then thawed and resuspended by pipetting or gentle vortexing in lysis buffer, repeating the process several times. Between cycles the sample is centrifuged, and the supernatant containing soluble protein is retained. Osmotic shock: This is a very gentle method that may be sufficient for the lysis of suspended mammalian or bacterial cells without the use of a detergent. The method, often combined with mechanical disruption, relies on changing from high to low osmotic medium, and is well-suited to applications in which the lysate is to be subsequently fractionated into subcellular components. Ultrasonication: This method of protein extraction is most frequently applied to cell suspensions. Cells are disrupted by high-frequency sound waves via a probe inserted in the sample. The sound waves generate a region of low pressure, causing disruption of the cell membranes. Mechanical methods: Proteins may be extracted from cells and tissues using various crude but effective "crushing and grinding" measures. For example, cell membranes may be disrupted by liquid shear forces using Dounce or Potter-Elvehjem homogenization. Tissues may be homogenized by chopping or mincing in chilled buffer using a Waring blender or Polytron® homogenizer. Tissues or cells may be frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle with alumina or sand. Rapid agitation of cells with fine glass beads disrupts cell walls, effective for most Gram positive and Gram negative bacteria. Enzymatic digestion: Enzymatic digestion: Enzymatic methods are frequently used when extracting proteins from bacteria, yeast, or eukaryotic cells embedded in fibrous tissues where cell membranes are surrounded by a robust protective structure. Cell lysis enzymes or cocktails such as Lysozyme, Mutanolysin, MetaPolyzyme, Lysonase, and Pronase can be used in combination with tissue digestion enzymes (i.e. Collagenase, Chondroitinase, Hyaloronidase) to dissolve or disrupt cell walls, coats, capsules, capsids, or other structures not easily sheared by mechanical methods alone. Enzymatic digestion can be followed by homogenization, sonication, or vigorous vortexing in a lysis buffer.

Additionally, because endogenous proteases and phosphatases may be liberated upon cell disruption and degrade the target molecule, the sample should be protected during cell disruption and subsequent purification using protease and phosphatase inhibitors to avoid uncontrolled loss of target.


Bacterial PE LB™ (9 Citations)

Bacterial PE LB&trade has been developed for the extraction of soluble proteins and inclusion bodies from bacterial cells. It is a proprietary improvement on the lysozyme based lysis, which allows extraction of soluble proteins and concurrent removal of nucleic acids (DNA & RNA) released during cell lysis. Bacterial PE LB&trade lysis eliminates viscosity build-up, allowing effective clarification with lower centrifugal force.

This kit is provided with an optional protocol for the formation of spheroplast and removal of lytic enzyme (Lysozyme) prior to lysis and extraction of the bacterial proteins. Bacterial PE LB&trade is based on organic buffering agents and utilizes a mild non-ionic detergent and a proprietary combination of various salts and agents to enhance extraction and stability of proteins. Depending on the application, additional agents such as reducing agents, chelating agent, and protease inhibitors cocktail may be added into Bacterial PE LB&trade. This reagent has been tested for use with several widely used bacteria including E. coli strains.

Bacterial PE LB&trade is compatible with most downstream applications including running various chromatography, gel electrophoresis applications, and protein folding procedures. Bacterial PE LB&trade is also compatible for protein estimation with NI&trade protein assay (Non-Interfering Protein Assay)&trade.

For bacterial lysis, the kits are suitable for extracting soluble proteins from approximately 30g wet cell pellets for every 100ml Bacterial PE LB&trade. When the kits are used for extracting soluble proteins from spheroplasts, they are suitable for approximately 9g wet cell pellets for every 25ml Bacterial Suspension Buffer supplied.

Bacterial PE LB&trade Formats

Complete Kits (Cat. # 786-176, 786-187, 786-188)

The complete kits feature Bacterial PE LB&trade, bacterial suspension buffer and PE LB&trade Lysozyme. The PE LB&trade Lysozyme is a proprietary mix of lysozyme, to break open cells, and DNase and RNase, to remove nucleic acids. The enzyme mix is ready-to-use and at concentrations for optimal enzymatic activity.

Bacterial PE LB&trade buffer only (Cat. # 786-177, 786-185, 786-186)

The buffer only option allows researchers to substitute the Bacterial PE LB&trade buffer into their existing protocols for improved lysis or for use in further downstream applications.

Bacterial PE LB&trade [2X] (Cat. # 786-189)

Bacterial PE LB&trade [2X] is a modified formulation of Bacterial PE LB&trade that allows half the volume of lysis buffer to be used while maintaining lysis rates. It is used for extraction of proteins that are expressed at low levels and can also be used when high concentrations of extracted proteins are desired.

Bacterial PE LB&trade in Phosphate Buffer (Cat. # 786-191)

Bacterial PE LB&trade in Phosphate buffer is a variation of Bacterial PE LB&trade buffers only in phosphate buffer. It has the same efficiency as Bacterial PE LB, however the resulting lysates can be used for protein labeling and coupling reactions that use primary amines.


Optimized cell lysis buffer for affinity-tagged protein purification:

  • Extract proteins from bacterial, yeast, mammalian, and baculovirus-infected cells
  • Fast, easy procedure&mdashrequires only a 10-min incubation
  • Mild, non-denaturing extraction helps preserve biological activity
  • Compatible with all IMAC resins, allowing quick purification of his-tagged proteins
  • Universal&mdashsuitable for any scale protein extraction & any tag (use with his-, GST-, FLAG- HA-, or other tagged proteins)
  • Efficient&mdashbetter than sonication or other cell lysis buffers

4. Microfabricated Platforms for Cell Lysis

Microfluidics is one of the emerging platforms for cell lysis on a micro scale. Microfluidics is the manipulation and handling of small volumes (nano- to picoliters) of liquid in microchannels. Due to the micro scale operation regime, microfluidics is well suited for application where the sample or sample volume is small. This lowers the cost of the analysis due to low consumption of reagents [46]. Microfluidics also enables integration of different modules (or operations) into one device. For example, cells can be lysed and the intracellular products can directly be post processed (PCR or DNA isolation for diagnostics) inside the same device [47,48]. Although there have been a number of reviews on cell lysis in the past 10 years [7,8,49], some of the recent developments in the field have not been reviewed. This review will focus on the recent developments from 2014 onwards and will briefly cover the developments from before, which have been extensively surveyed. Some of the macro scale techniques have been implemented in microfabricated devices for cell lysis. Techniques such as electrical lysis methods are applicable only in the micro scale. Microfluidic lysis technology can be broadly classified into six types. They include mechanical lysis, thermal lysis, chemical lysis, optical lysis, acoustic lysis and electrical lysis.

4.1. Mechanical Lysis

Mechanical lysis in microfluidics involves physically disrupting the cell membrane using shear or frictional forces and compressive stresses. Berasaluce et al. [50] developed a miniaturized bead beating based method to lyse large cell volumes. Zirconium/silica beads were placed inside a cell lysis chamber along with a permanent magnet and actuation of an external magnetic field caused the motion of the beads inside the chamber. Figure 7 shows the various components and device assembled for cell lysis. Staphylococcus epidermidis cells were used in this study and they studied the effect of bead size, volume, flow rate and surfactant (Tween-20) on lysing efficiency. They found the optimum parameters achieved a 43% higher yield efficiency at a flow rate of 60 μL/min compared to off chip bead beating system.

Miniaturized bead beading cell lysis system: (a) various components: (1) inlet (2) outlet (3) stirring magnet (4) zirconia/silica beads (5) bead weir (6) rotating magnet and (7) electric motor coupling and (b) image of the device for lysis. Reproduced with permission from [50].

Pham et al. [51] have recently used nanotechnology to fabricate black silicon nano pillars to lyse erythrocytes in about 3 min. They fabricated these nanopillar with

12 nm tip diameter and 600 nm tall on silicon substrate using reactive ion etching technology. The authors showed that the interaction of erythrocytes cultured on nanopillar arrays causes stress induced cell deformation, rupture and lysis in about 3 min. Figure 8 shows the interaction of erythrocytes with the nanostructures.

Cell lysis using nano pillars: (a,b) top and side view of the cells interacting with the nanopillars and (c) confocal laser scanning microscopy pictures of intact, deformed and ruptured cells. Reproduced with permission from [51].

Mechanical lysis has been demonstrated by using nano-scale barb [52]. When cells are forced through small opening, high shear forces cause rupture of the cell membrane. Similar principle has been used here where “nanoknives” were fabricated in the wall of microchannels by using modified deep reactive ion etching (DRIE). Distance between these sharp edges was 0.35 μm and width of the channel was 3 μm. The lysis section of this device consisted of an array of these “nanoknives” patterned on a microchannel as shown in Figure 9 b. Human promyelocytic leukemia cells (HL-60) were used to pass through this section at sufficient velocity. The addition of this “nanoknives” pattern increased the amount of lysis. This device was used to extract protein from inside the cell. It has been estimated that as much as 99% of the cell was lysed but, only 6% protein was released.

Mechanical lysis using nanoscale barbs: (a) microfluidic device showing different inlets and outlet channels (b) schematic of the barbs (c) deep reactive ion etching (DRIE) fabricated nano-knives (d) magnified image of nano-knives patterned using DRIE technique and (e) dimensions of the nano-knives used for cell lysis. Reproduced with permission from [52].

Alternatively, mechanical impingement through collision has also been used to lyse in the microscale [53,54,55]. Cells were suspended in solution with glass beads and placed on the microfluidic compact disc (CD) device, which was then set to rotate at a very high velocity. The centrifugal force generated by the rotation, causes collision and friction between cells and beads, which results in cell lysis. Various kinds of cells including mammalian, bacteria and yeast have been lysed using this technique.

Though the efficiency of the mechanical lysis is very high, these disruption methods have some drawbacks in microscale application. Fabrication of these devices is complex as well as expensive and collecting the target materials from a complex mixture is very difficult.

4.2. Thermal Lysis

In thermal lysis, heat is supplied to the cells to denature the membrane proteins and lyse the cells. One advantage of thermal lysis is the easy integration of microfluidic devices such as polymerase chain reaction (PCR). The thermal lysis can be performed in such devices with no additional modification. The cells are generally heated above 90 ଌ and the intracellular products are cycled through different temperatures for example in a PCR device. Tsougeni et al. [56] fabricated a microfluidic device which can capture and lyse cells. They used thermal lysis at 95 ଌ for 10 min to capture and lyse bacteria. Nanostructures were fabricated in poly(methyl methacrylate) using lithography and plasma etching technique. Microfluidic PCR devices which have incorporated thermal cell lysis [57,58,59] consist of a glass chamber and a resistive heater to heat the chamber.

In general, thermal lysis is effective in a microfluidic platform, however, these devices are not suitable for sample preparation where the sample is of a large volume and cells have to be lysed from a continuous flow [29]. However, cells have to be treated with lysozyme in order to break the cell wall and make bacteria protoplast. The addition of this lysozyme is time consuming and requires complex structures. Moreover, preserving the enzyme within the device becomes problematic when the device has to be used for a long period of time. Higher lysis time and elevated power consumption are other drawbacks of this method.

4.3. Chemical Lysis

Chemical lysis methods use chemical reagents such as surfactants, lysis buffers and enzymes to solubilize lipids and proteins in the cell membrane to create pores and lyse cells. Although chemical and enzymatic methods are categorized separately in macro scale method, these two techniques are incorporated in the same group for micro scale cell lysis techniques. Buser et al. [60] lysed gram-positive bacteria (Staphylococcus aureus) and RNA virus (respiratory syncytial virus) using a dried enzyme mixture (achromopeptidase). They were able to lyse in less than a minute and then used a disposable chemical heater to deactivate the lysis enzyme. They were able to amplify (off-chip) the lysate without purification and showed the proof of principle for a point of care device for diagnostics.

Kashyap et al. [61] developed a microfluidic probe for selective local lysis of adherent cells (

300 cells) for nucleic acid analysis. Hall et al. [62] used a device for cell lysis experiment, which had two supply wells and a pressure well. Mixing of cell and lysis solution was controlled by adjusting the pressure of the wells. Three different types of solution were used—Solution A containing only SDS (detergent based reagent), Solution B containing surfactant, Triton X-100, Tween-20 with enzyme such as lysozyme, protease, proteinase K and Solution C containing an antibiotic named polymyxin B. Gram-negative and gram-positive bacteria were used for lysis. It was concluded that detergent alone was not suitable for lysis, while Solution B, a mixture of chemical surfactants and biological reagents, can disintegrate the cell membrane and lyse various kinds of bacteria. However, polymyxin B can be potentially used in microfluidic cell lysis platform only for gram-negative bacteria.

Kim et al. [63] also developed a microfluidic device with two inlets and outlets in order to develop an optimal lysis reagent for gram-negative bacteria. Heo et al. [64] demonstrated a microfluidic based bioreactor which was capable of entrapping E. coli by using hydrogel patches. Then the immobilized E. coli was lysed by using SDS as it can penetrate hydrogel. Cell lysis was accomplished within 20 min. This device was capable of cell lysis using only SDS, however, the previous one could not due to lower exposure time in chemical environment. In another study, Sethu et al. [65] also developed a microfluidic chip ( Figure 10 ) to lyse Erythrocyte in order to isolate Leukocyte. One hundred-percent recovery was possible within 40 s. The device consists of three inlet reservoirs and one outlet reservoir. One inlet was used to flow the entire blood. Second inlet was used for lysis buffer containing mainly aluminum oxide and two side channels were connected with this inlet which converged to direct the entire blood into a narrow stream. This increases the surface contact between the lysis buffer and the cells. The mixture of cells and lysis buffer was then run through a long channel with a number of “U” turns to enhance the buffer. Finally, third inlet was used to flow the phosphate buffer in order to dilute the sample for restoring the physiological concentration [66,67].

Schematic of a simple chamber and serpentine microfluidic channel for chemical lysis. Reproduced with permission from [65].

Even though chemical lysis method is widely used in many microfluidic devices, this method requires an additional time consuming step for reagents delivery. Therefore, complex microfluidics structures including injection channels and micro-mixers to homogenize the samples are needed [66,68]. After lysis, these reagents might interfere with downstream assay as it is very hard to separate the target molecules [69]. In addition, storage of these reagents is a problem which is why the device cannot be used for long time.

4.4. Optical Lysis

Optical lysis of cells involves the use of lasers and optically induced dielectrophoresis (ODEP) techniques to break open the cell membrane. In laser lysis, a shock wave created by a cavitation bubble, lysis the cell membrane. A focused laser pulse at the cell solution interface creates this cavitation bubble. In ODEP, a conductive electrode and a photoconductive layer (for example amorphous silicon) are formed on the top surface of glass slide. A non-uniform electric field is generated by shining light on the photoconductive layer which then generates a transmembrane potential across the cell membrane disrupting the cell membrane. Huang et al. [70] developed an optically induced cell lysis microfluidic chip for lysing HEK293T cells and extracting intact nucleus. They report cell lysis and nucleus separation efficiency as 78% and 80% respectively using this device.

Kremer et al. [71] lysed cells using an opto-electrical setup. They were able to lyse cells selected based on shape of the cell. They used ODEP to lyse red blood cells in a mixture of red and white blood cells. They developed a method that enabled shape-selectivity such that cells with a different geometry will lyse in a mixture of cell types. The cell with a different shape induces a non-uniform electric field which is used for lysis. Figure 11 shows the schematic of the lysis chip and lysis of differently shaped cells.

Optical cell lysis device: (a) cell lysis chip using optically induced dielectrophoresis (ODEP) (bd) cell lysis of red blood cells in a mixture of white and red blood cells and (eg) lysis of red blood cells in a mixture of red blood cells and trypanosomes. Reproduced with permission from [71].

Use of laser light to induce lysis has also been attempted in microfluidic devices. In one instance, optical lysis was induced by application of a nanosecond 532 nm laser pulse [72] which generates a microplasma locally. The plasma collapses causing cavitation, bubble expansion and its collapse as described in previous section are the main reason for a laser induced cell lysis. Various types of cell lines such as rat basophilic leukemia (RBL) [73], rat-kangaroo (Potorous tridactylis) epithelial kidney cells (PtK2) [74], and murine interleukin-3 dependent pro-B (BAF-3) [75] have been lysed by using this laser induced method. However, all these experiments had been done for single cell analysis. It has been found that when laser based lysis was incorporated with polydimethylsiloxane (PDMS) microchannel efficiency of lysis decreased [75]. It was suggested that this may be due to the deformation of PDMS walls which dissipates the mechanical energy from the bubble collapse. For that reason, high energy was required.

Ultraviolet (UV) light array combined with titanium oxide has been used to lyse the cell [76]. Titanium oxide possesses photolytic properties and excitation energy that falls within UV range. When titanium oxides are excited with UV light array, electrons in the valence band are excited to conduct ion band which results in electron–hole pairs. In aqueous environment, these electron–hole pairs react with surrounding molecules and generate free radicals such as OH, O and O2 − . These react with cell membrane and lyse the cell. E. coli cells were lysed with the above technique. A primary disadvantage of ultraviolet lysis was that the time required to lyse the cell was very high (45 min).

4.5. Acoustic Lysis

In acoustic lysis, a high energy sound wave is generated which is used for cell lysis. This surface acoustic wave (SAW) is produced on a piezoelectric substrate. An inter-digitated transducer (IDT) can be used to produce a SAW electrically with the wave propagating on the surface away from it. Taller et al. [77] have used on chip surface acoustic wave lysis for detecting exosomal RNA for pancreatic cancer study. They achieved a lysis rate of 38% using this technique. Figure 12 shows the fabricated device with the SAW transducer.

Surface acoustic wave (SAW) lysis microfluidic device: (a) assembly of device and (b,c) as fabricated device with liquid inlet and outlet for exosome lysis. Reproduced with permission from [77].

They report that the lysis of exosomes is possible due to the effects of acoustic radiation force and dielectric force acting on small particles [78,79]. The SAW device was fabricated using standard photolithography technology. Twenty pairs of titanium aluminum electrodes were patterned on top of piezoelectric lithium niobate substrate to form a single phase unidirectional SAW transducer. This transducer can generate SAW in only one direction. Raw media was exposed to SAW for 30 s at 1 W of power for lysing. The authors report that a lysis efficiency of 38% achieved using this method was sufficient for obtaining enough exosome RNA for detection.

Marentis et al. [80] lysed the eukaryotic cell as well as bacteria by using sonication. This device consists of a microfluidic channel with integrated transducer. The channel was made on glass substrate and piezoelectric transducer was made by depositing zinc-oxide and gold on quartz substrate. The transducers were driven by a sinusoidal source in the 360-MHz range. Eighty-percent lysis of HL-60 and 50% lysis of Bacillus Subtilis spores were obtained by using this device. The temperature rise due to sonication was moderated by using ice pack and cold finger. Ultrasonic horn tip and liquid region are coupled in a microfluidic chip by increasing fluidic pressure in order to increase the efficiency of lysis [81].

Reboud et al. [82] have developed a disposable microfluidic chip to detect the rodent malaria parasite Plasmodium berghei in blood. They used SAW to lyse the red blood cells and parasitic cells in a drop of blood. They report a cell lysis efficiency of more than 99.8% using their device. Xueyong et al. [83] have fabricated a SAW microfluidic device which can lyse red blood cells with high efficiency (95%).

However, sonication has limitations such as generation of heat, complex mechanism as well as expensive fabrication process. Due to this excessive heat generation denaturation of protein and excessive diffusion of the cell contents have been observed [8,84]. To reduce the operation time, cells were first treated with some weak detergent such as digitonin [8,85] before ultrasonic exposure. Digitonin weakened the cell membrane and facilitated lysis.

4.6. Electrical Lysis

In electrical method, cells are lysed by exposing them to a strong electric field. An electric field is applied across the cell membrane which creates a transmembrane potential. A potential higher than the threshold potential is required to form pores in the cell membrane. If the value of the potential is lower than the threshold potential, the pores can be resealed by the cell. On the other hand, a high enough potential can completely disintegrate the cell. At such high voltages, it is found that the electric field does not have any effect on the intracellular components [86]. Electric field is the critical parameter to lyse the cell. As higher electric field is required for cell lysis, high voltage generator is required in order to generate this high electric field in macroscale. Thus, this method is not common in macroscale. However, in microscale due to small size of the devices, higher electric field can be obtained at lower voltage. For this reason and as a method for fast and reagentless procedure of lysis, electrical lysis has achieved substantial popularity in microfluidic community.

Ameri et al. [87] used a direct current (DC) source to lyse cells in a microfluidic chip. Figure 13 shows the fabrication and working principle of their chip. Their device consists of a glass slide coated with indium tin oxide coating patterned for electrodes. The 6400-Microwell arrays are fabricated using SU-8 polymer by photolithography technique. Inlet and outlet channels are created using PDMS polymer and is sealed using a glass slide with ITO electrode for impedance measurement. Red blood cells (10 7 cells/mL) are flown through the device at 20 μL/min and dielectrophoresis (DEP) is used to immobilize the cells into the microarray. A DC voltage of 2 V for 10 s was applied to the cell for lysis. The lysis process was monitored using impedance measurement before and after lysis and a decrease in impedance suggested a complete lysis of cells. They report a lysis efficiency of 87% in their device. The authors proposed a device for cell lysis by electric fields and optical free monitoring of the lysis process on a microfluidic platform which could have potential use in the medical diagnostic field.

Electrical cell lysis device: (a) fabrication protocol of the device (b) working principle of the device and (c) microfluidic device used in the study for lysing red blood cells. Reproduced with permission from [87].

Jiang et al. [88] developed a low cost microfluidic device for cell lysis using electric fields. They applied a 10 V square pulse to lyse cells at 50% efficiency. They report a device which had the capability to lyse cells at a much lower voltage compared to a commercially available electropolator device which operated at 1000 V to lyse 200 μL of PK15 cells. They observed bubble formation in their device during cell lysis due to joule heating effect. De Lange et al. [89] have lysed cells in droplets using electric fields. They demonstrated a robust new technique for detergent free cell lysis in droplets. In their device, electric field was applied to lyse bacteria immediately before merging the cell stream with lysozyme and encapsulating the mixture in droplets. They report that with lysozyme alone the lysis efficiency is poor (less than 50%) but when combined with electric fields they were able to obtain up to 90% cell lysis efficiency. Figure 14 shows their microfluidic device for cell lysis in droplets. The authors suggest that their device could be used in applications where use of cell lysis detergents could hinder the cell analysis such as binding assays or studying the chemical activity of proteins and in mass spectroscopy studies where chemical lysis agents can hamper the results.

Electrical cell lysis microfluidic device: (A) schematic of the electrical lysis and coflow droplet generation microfluidic chip (B) actual image of the droplet generation part and (C) complete electrical lysis with electroporation channels. Reproduced with permission from [89].

Escobedo et al. [90] showed electrical lysis of cells inside a microfluidic chip using a hand held corona device. They were able to lyse baby hamster kidney cells (BHK), enhanced green fluorescent protein human-CP cells (eGFP HCP) 116 and non-adherent K562 leukemia cells completely inside a microfluidic channel. A metal electrode was embedded inside the channel which was used to discharge 10 to 30 kV to lyse the cells in less than 300 ms. Lysis was assessed by observing before and after images of cells using bright field and high speed microscope and also by cell-viability fluorescence probes. They also report no bubble formation during lysis indicating no joule heating effect thereby making this method suitable for analyzing sensitive proteins and intracellular components. Figure 15 shows the setup and results of the study.

Electrical lysis through handheld plasma device: (a) schematic of the device. Cells were lysed using a hand held corona device by applying electric field at the inlet of the device (b) bright field and fluorescent images of before and after of lysis of K562 cells. Reproduced with permission from [90].

Besant et al. [91] detected mRNA molecules of E. coli by electrochemical lysis technique. They applied a potential of 20 V, which initiated the cell lysis by producing hydroxide ions from water at cathode to break down bacterial membranes. The sensor electrodes were placed 50 μm away which was enough to detect the mRNA molecules in 10 min. They reported lysis and detection of E. coli mRNA at concentrations as low as 0.4 CFU/μL in 2 min which was relevant for clinical application in both sensitivity and time.

Gabardo et al. [92] developed a low cost and easy method to fabricate multi-scale 3D electrodes that could be used for bacterial lysis using a combination of electrical and electrochemical means. These micron-sized electrodes can be rapidly prototyped using craft cutting, polymer induced wrinkling and electro-deposition techniques. They report that these tunable electrodes performed better as compared to lithographically prepared electrodes. They were able to successfully extract nucleic acids extracted from lysed bacteria on a microfluidic platform. They reported 95% lysis efficiency at 4 V using their electrodes. Figure 16 shows the device and electrode structures.

Bacterial lysis device: (a) schematic of the lysis device (b) scanning electron micrographs of: (i) planar (ii) wrinkled and (iii) electrodeposited electrodes (c) cyclic voltammetry scan of the electrodes. Reproduced with permission from [92].

Li et al. [93] developed a double nano-electrode electrical cell lysis device to lyse single neuronal cells. Similarly, Wassermann et al. [94] showed cell specific lysis of up to 75% of the total human blood cells using SiO2 passivated electrical cell lysis electrodes at an applied voltage of 8� V. Ma et al. [95] reported a 10�-fold increase in mRNA extracted from M. smegmatis using electrical lysis in a microfluidic platform as compared to a commercial bead beading instrument. They used a 4000� V/cm field intensity to lyse the bacteria with long pulses (5 s). They report that their device can be effective for mRNA release from hard to lyse cells.

Islam et al. [96] showed the proof of concept of a simple microfluidic device for electrical lysis of larger volumes of sample. They used a nanoporous membrane sandwiched between two microfluidic channels to trap and lyse E. coli bacteria by applying 300 V. They report a lysis efficiency of 90% in less than 3 min. Figure 17 shows the schematic of the device used for lysis in their study.

Electrical cell lysis microfluidic device: (a) schematic of cell lysis device and (b) experimental setup. Reproduced with permission from [96].

Different types of voltages such as alternating current (AC) [97,98], DC pulses [99,100,101] and continuous DC voltages [102] have been used in order to lyse the cells. Along with electric field, exposure time of cells within that electric field is also an important parameter for cell lysis. It has been found that cells can be lysed by using higher electric field for short period of time as well as lower electric field for long period of time [103]. For that reason, AC and DC pulses of a higher electric field are needed as compared to a continuous DC electric field. As the electric field depends on the distance between the electrodes, microfabricated electrodes have been used during AC or DC pulses. An overview of different electrical lysis devices and the characteristics of the designed system is presented in Table 4 .

Table 4

Different electrical lysis devices used for cell lysis.

ReferenceSpeciesType of CellCell Size (μm)ElectrodeType of Voltage (AC/DC)Lysing Voltage (V)
[104]HumanHT-2910GoldAC8.5
[97]HumanA43110GoldAC20
[98]-FITC-BSA laden vesicle50ITOAC5
[99]-Leukocytes-3DDC pulse10
[100]HumanRed blood cells6𠄸Pt wireDC/AC30�
[105]BacteriaE. coli-GoldDC pulse50
[106]HamsterCHO10�Pt wireDC pulse1200
[106]BacteriaE. coli Pt wireDC930
[102]HumanRed blood cells6𠄸Pt wireDC50
[87]HumanRed blood cells6𠄸ITODC2
[96]BacteriaE. coli-Pt wireDC300

Lu et al. [104] developed a microfluidic electroporation platform in order to lyse human HT-29 cell. Microfabricated saw-tooth electrode array was used in order to intensify the electric field periodically along the channel. Seventy-four-percent efficiency was obtained for an operational voltage of 8.5 V. However, this mode of lysis is not suitable for bacteria due their sizes and shapes. Compared to mammalian cell, high electric field and longer exposure is needed to lyse bacteria. Rosa [105] developed a chip to lyse bacteria consisting of an array of circular gold electrodes. DC pulses were used and lysis with 17% efficiency was achieved by using an operational voltage 300 V. This efficiency was increased up to 80% after adding enzyme with cell solution. In 2006, Wang et al. [107] proposed application of continuous DC voltage along the channel for cell lysis. The device consists of a single channel with uniform depth and variable width. Since the electric field is inversely proportional to width of the channel, high electric field can be obtained at the narrow section of the channel. Thus, lysis occurs into a predetermined portion of the device. Exposure time of the cell to the electric field can be tuned by changing the length of this narrow section. The configuration of the device was optimized and lysis of complete E. coli bacteria was possible at 930 V. Complete disintegration of cell membrane was observed when the electric field was higher than 1500 V/cm. This device was very simple and did not need any microfabricated electrodes. Pt wires were used as electrodes. Only a power generator was needed to operate it. However, bubble generation and Joule heating issue could not be completely eliminated. Similar kind of device was used by Lee [102] where the length and width of the narrow section was modified in order to lyse mammalian cell. Bao et al. [108] also developed a device to lyse E. coli by using DC pulses. Release of intracellular materials was observed when the electric field was higher than 1000 V/cm.

In conclusion, electrical method offers a simple, fast and reagent less lysis procedure to lyse various kinds of cells. This method is also suitable for selective lysis and is compatible with other downstream assays such as amplification and separation. Although requirement of high voltage is a problem in this procedure, it can be overcome by decreasing the gap between electrodes through microfabrication. However, heat generation and formation of bubble is a major problem for electric lysis method.

4.7. Comparison of Different Microfluidic Technologies for Cell Lysis

Various microfluidic technologies for cell lysis are compared in Table 5 . The advantages and disadvantages of different methods are listed for each technique.

Table 5

Comparison of different microfluidic lysis methods. Cell lysis efficiency was determined by averaging the lysis efficiencies from the references cited. Low: 0%�% Medium: 50%�% High: 80%�%.


Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency.

This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.


How to Make a Cell Lysis Solution

The precise components and procedure required for making a cell lysis solution depends on several factors, including the type of cells and the objective of the experiment. This BiologyWise article explains how to make cell lysis solution with respect to the major ingredients, and how they vary with different experimental setups.

The precise components and procedure required for making a cell lysis solution depends on several factors, including the type of cells and the objective of the experiment. This BiologyWise article explains how to make cell lysis solution with respect to the major ingredients, and how they vary with different experimental setups.

Sodium dodecyl sulfate (SDS), the most commonly used detergent for cell lysis and protein denaturation, is also widely used in several soaps, shampoos, laundry detergents, and toothpastes.

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A cell lysis solution is a detergent-based buffer solution used to break open the desired cells and further isolate a particular cellular component of interest. It is also referred to as a cell lysis buffer or simply, lysis buffer. This process of lysing cells using chemical agents is termed as chemical disruption.

Apart from detergent and buffering agents, additional agents are added that aid the lysing process, eliminate unwanted cellular components, and/or protect the desired cellular component. These additions depend on the cell type involved, the cellular component to be studied, and the precise techniques that need to be performed on the lysate.

Given below is the basic procedure to prepare a cell lysis solution, followed by a description of the components required to prepare the solutions, and their variations with respect to the commonly followed protocols for different experiments.

Procedure to Make a Cell Lysis Solution

Given below is the procedure to prepare a lysis solution containing 10mM Tris-HCl buffer, 1mM EDTA as the chelating agent, and 0.5% SDS as the detergent.

Dissolve 121 g Tris-HCL (molecular weight = 157.60 g) in 800 ml distilled water, adjust the pH to 8 using HCl solution, and make up the volume to 1 L using distilled water.

Step 2: Preparation of 500 ml of 0.5 M EDTA stock solution

Dissolve 93.0 g of EDTA [EDTA.Na2.2H2O] (molecular weight = 372.24 g)] in 400 ml of distilled water, add 10 g (approx.) NaOH pellet to adjust pH to 8, and make up the volume to 500 ml using distilled water.

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Step 3: Preparation of 10% SDS stock solution

Dissolve 10 g of SDS in 90 ml distilled water, and make up the volume to 100 ml using distilled water.

Step 4: Preparation of 500 ml of the Tris-EDTA SDS lysis buffer

Choosing the Reagents for a Cell Lysis Buffer

All cell lysis solutions are prepared using a suitable buffer solution, so as to maintain the appropriate pH. Disruption of the cells will disturb the internal pH of the cell, which alters the structural integrity of proteins and other macromolecules. In order to avoid this, generally a buffer solution adjusted at a pH equal to the normal intracellular pH is used.

A buffer solution comprises a weak base and its conjugate acid, or a weak acid and its conjugate base. Each buffering agent can work as an effective buffer only within a specific pH range called the buffer range. The choice of buffering agent depends on this buffer range. Generally, the buffers are used at a concentration of 10 – 50 mM and are adjusted to pH 7.4 – 8.

The two commonly used buffers for making cell lysis solutions, and their buffer range are as follows:

Cell membranes are made up of phospholipid layers which are held together through polar interactions. A detergent serves as an emulsifier and disrupts these polar interactions. It also forms complexes with the lipid molecules and protein molecules embedded in the membrane, and precipitates them out of the solution.

Detergents can be grouped as ionic or non-ionic, depending on the nature of the hydrophilic group, or as mild or strong depending on their ability to solubilize membranes and proteins. Ionic detergents with positively charged groups are termed cationic detergents, and those with negatively charged groups are termed anionic detergents.

Mild detergents are used when cells need to be lysed for purifying and/or studying cellular proteins, so as to avoid denaturation of the desired proteins. On the other hand, strong detergents that denature cellular proteins are used while lysing cells for DNA isolation.

Given below are some of the commonly used detergents, their application as cell lysing agents, and usual concentrations in which they are used for cell lysis.

1) SDS (sodium dodecyl sulfate) [anionic]

General use: DNA isolation from any cell type, protein isolation under denaturing conditions
Concentration: 1 – 10 %

2) Sodium Deoxycholate [anionic]

General use: Purification of membrane proteins
Concentration: 0.5 %

3) CTAB (cetyltrimethylammonium bromide) [cationic]

1) NP-40 (nonyl phenoxypolyethoxylethanol)

General use: Nuclei isolation
Concentration: 0.1 – 1.0 %

2) Triton X-100

General use: Purification of membrane proteins, DNA isolation
Concentration: 0.1 – 5.0 %

3) Polysorbate 20

General use: Purification of membrane proteins, immunoprecipitation
Concentration: 0.05 – 0.5 %

Role: The chelating agent is a chemical that sequesters divalent cations, like Mg ++ , and Ca ++ , which are required for membrane stability. Due to such chelation, these cations are no longer associated with the membrane, thereby, weakening it. Moreover, the lysing of membrane results in exposing the nucleic acids to nucleases, which are otherwise sequestered inside the lysosomes. These nucleases are inactive in the absence of divalent ions. Thus, chelating agents protect the DNA and RNA molecules from degradation by these nucleases.

Osmotic Stabilizers: Glucose, sucrose, sorbitol, and glycerol may be added for osmotic stability, and preventing the cellular components from osmotic shock that may occur during the sudden rupturing of cell membrane. In addition, they also help stabilize lysosomal membranes, thereby, reducing the release of degradative enzymes from the lysosomal lumen.

Salts: Disruption of cells and the release of cellular components into the medium, may alter the ionic strength of the medium. In order to deal with this and maintain ionic strength, salts, like sodium chloride [NaCl], potassium chloride [KCl], and ammonium sulfate [(NH4)2SO4] may be added.

Enzymes: Depending on the target macromolecules to be isolated from cells, lytic enzymes may be added to either protect them from the action of other enzymes, or degrade the remaining macromolecules that may contaminate the lysate.

Cell lysis solutions are used for the chemical disruption of cells, and may also be used in a combination with other methods, like homogenization, sonication, grinding, freeze-thaw techniques, etc. This is especially useful for lysis of fungal cells and plant cells, since they have strong cell walls around the cell membranes.

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Lysis and Extraction Systems

An array of reagents and accessories for extraction of proteins from biological samples. Depending on applications and biological sample type, researchers are offered the options to choose from one or more of the following lysis and protein extraction systems. These protein extraction systems also offer researcher the option to select buffering agents either amine based, non-amine or phosphate buffering agents. These lysis systems are designed to cover the most extensive range of applications in protein research.

The Protein Extraction and Lysis Buffer systems (PE LB&trade series) utilize a mild detergent, ensuring efficient protein recovery while maintaining the biological activity of the proteins. The solubilized proteins are suitable for enzyme assays, electrophoresis, ELISA, Western blot, folding studies, chromatographic studies, and many other downstream applications. The PE LB&trade systems offer a wide selection of lysis buffers for extraction of proteins from bacteria, insects, mammalian cell cultures, mammalian tissues, and yeast.

PopLysis&trade protein extraction systems utilize a cocktail of membrane solubilizing detergents and are optimized to rapidly solubilize and &ldquopop open&rdquo membrane structures, allowing cellular proteins to spill out into lysis solution. The cellular proteins are stable in PopLysis&trade buffer and suitable for downstream applications such as enzyme assays, chromatography studies, ELISA, Western blot, protein folding studies, and gel electrophoresis. These systems are also suitable for high throughput applications such as screening recombinant proteins. PopLysis&trade buffers contain amine-free buffering agents making them suitable for applications that require proteins in amine free buffers. PopLysis&trade buffer systems are optimized kits for lysis and extraction of proteins from bacteria, yeast, neurons, mammalian cells, mammalian tissue, and insect cells.

FOCUS&trade Proteome Kits are optimized for the preparation of total protein for downstream screening and protein discovery. These kits have been optimized for the isolation of several protein types including soluble, insoluble, membrane, cytoplasmic, nuclear, signal, phosphoproteins and glycoproteins. These kits are simple to use, save time, improve the quality of protein analysis and improve the likelihood of novel protein discovery. FOCUS&trade Proteome Kits are suitable for the analysis of proteins using 1D and 2D electrophoresis, mass-spectrometry, and other sensitive biochemical techniques. Each kit is specifically designed for protein isolation from either mammalian cells, mammalian tissues, bacteria, insects, plants, yeast, and other biological samples.

Other lysis buffer offerings include the extensively cited RIPA lysis buffer, and a modified mild version Mild-RIPA lysis and extraction buffer that does not contain SDS detergent. Inclusion bodies solubilization buffers, IBS&trade and HP- IBS&trade Buffers. A kit for Total Protein Extraction (TPE&trade) for the extraction of total proteins from cells and tissues for electrophoresis and other applications. RBC lysis buffer specifically designed for lysis of red blood cells.

Accessories are also offered to assist with protein extraction and isolation procedures which include our EZ Grind&trade and Molecular Grinding Resin&trade, sample grinding tools, they are available either as single use tubes with matching pestles or just grinding resin with or without matching tubes and pestles. We also offer ProteaseArrest&trade, a complete range of protease Inhibitor cocktail solutions. PhosphataseArrest, for phosphatase sensitive protein extraction applications. Metal chelators (EDTA & EGTA), reducing agents, various salts (both monovalent and divalent), lytic enzymes, denaturing agents, proteomic detergents, and other additives for protein research.


Conclusions

It is widely acknowledged that bias exists in 16S rRNA studies describing microbiota profiles and that no currently available method is able to perfectly describe the community being analysed [10]. However, an understanding of how the choice of laboratory methods affects the results of such studies is important in order to accurately interpret the results and make valid comparisons between different studies. Although we were able to identify significant differences in DNA yield and diversity between the different methods used in this study, the effects of this were much smaller than those due to the sample and did not alter the grouping of extracts by hierarchical clustering and principal coordinate analysis. However, since there was an observable effect of lysis method on microbiota composition, we recommend that the same method is used within a study to reduce the risk of introducing a differential bias. Furthermore, comparisons between studies using different lysis methods should be made with care, but will likely be of much smaller magnitude than differences caused by the choice of extraction kit and 16S rRNA primers [12]. Additionally, studies with a focus on the abundance of a particular bacterial species should include additional techniques such as qPCR to confidently identify any differences between groups.


Watch the video: Bacterial Cell Lysis Tutorial for Protein Extraction using ProBlock Protease Inhibitors (May 2022).