Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool for analyzing the proteome of bacterial cells. To prepare bacterial samples for 2-D PAGE, several steps are required to extract and solubilize the proteins.
The first step is cell lysis, which can be achieved by mechanical disruption, such as sonication or bead beating, or by chemical lysis using detergents or chaotropic agents. The choice of lysis method depends on the type of bacteria and the properties of the proteins to be extracted.
Once the cells are lysed, the protein extract can be further purified and solubilized using different techniques, such as ammonium sulfate precipitation, phenol extraction, or organic solvent extraction. These methods can remove contaminants and improve protein solubility, which is important for 2-D PAGE.
After the protein extract is purified and solubilized, it can be separated using 2-D PAGE, which involves two steps of electrophoresis. In the first dimension, proteins are separated based on their isoelectric point using isoelectric focusing (IEF). In the second dimension, proteins are separated based on their molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins can then be visualized by staining with a protein dye, such as Coomassie Blue or silver stain.
To identify the proteins on the gel, different methods can be used, such as peptide mass fingerprinting or tandem mass spectrometry. These methods can provide information on the identity, quantity, and post-translational modifications of the proteins.
1 General Solubilization Protocol
It is crucial that all bacteria be disrupted, so that the lysis buffer gains access to all proteins. In studies where multiple extractions with different chemical reagents are employed sequentially, it will mask the result if more and more bacteria are disrupted during the procedure. The best method for disruption is dependent on the type of bacteria.
In most cases, disruption by sonication will do, but it may be necessary to add lysosyme to break down cell walls. It is advantageous to perform the disruption in SDS or in lysis buffer containing thiourea, in which most proteases are denatured. If prolonged manipulation, such as fractionation by different steps of centrifugation, must be performed, protease inhibitors should be added (see Note 9). In the procedure described below, the bacteria are sonicated in 2% SDS, 65 mM DTE, and boiled in order to enhance the protein solubilization in general. It has been suggested that SDS used for presolubilization does not interfere with first-dimensional electrophoretic separation because it forms micelles with the nonionic detergent of the lysis buffer and migrates out of the strip. Still, the amount of SDS should be kept low compared to the amount of detergent in the lysis buffer.
The protocol below describes the solubilization of proteins from pelleted bacteria. If proteins have been precipitated as a purification step, the lysis buffer should be added directly (step H) in the highest possible amount.
A. Start out with an appropriate amount of bacteria as pellet.
B. Add four times the pellet volume of 2% SDS, 65 mM DTE.
C. Sonicate three times for 2–20 s depending on sample size.
D. Spin briefly to collect the sample.
E. Resuspend any pellet that may have formed.
F. Boil for 5 min.
G. Allow the sample to cool.
H. Add 8 vols of lysis buffer to one vol of extract
I. Sonicate three times for 5 s, cool between sonications.
J. Leave the sample on a rocking table for 30 min.
K. Spin at 20,000g for 15 min and collect the supernatant.
L. Assess the protein concentration.
M. Run first dimension immediately or store the sample at –70°C for several months.
2 Sample Purification
Common contaminants in 2-D PAGE studies are salts, small ionic compounds, polysaccharides, nucleic acids, and lipids. Salt is the most likely reason if bad first-dimensional focusing is observed. Enhanced conductivity and water migration in the strip due to high concentrations of salts will cause horizontal streaks. The concentration of salt should be below 10 mM when samples are loaded by strip rehydration. Small charged substances may likewise disturb the isoelectric focusing. Polysaccharides may clog the gel of the strip and may complex proteins by electrostatic interactions. Lipids may also clog the gel but are mainly a problem due to complexing of hydrophobic proteins and binding of detergent. Nucleic acids may clog the gel, bind proteins through electrostatic interactions, and cause streaking, especially in silver staining.
Dialysis and precipitation are straightforward and effective ways to reduce the concentration of salt and small ionic compounds to an acceptable level. Dialysis causes a minimal loss of sample, but requires relative large volumes of solute and is rather time consuming. Spin dialysis using, for instance, Amicon Ultra from Millipore is faster and requires no extra volume of solute, but protein may be lost by adsorption onto the dialysis membrane. Precipitation may also be used to remove polysaccharides and to some extent lipids. Large polysaccharides can be removed by ultracentrifugation. If lipids are causing major problems, the amount and nature of detergent must be optimized for the particular sample. High amounts of nucleic acids may require treatment with DNase/RNase.
The presence of proteases is likely to be a problem during sample purification, and in that case protease inhibitors must be added. It must be stressed that sample purification preceding addition of lysis buffer should be carried out only if necessary and not as a standard part of the sample preparation.
2.1 Precipitation
Precipitation is very efficient for removal of most contaminants, including salts, but no precipitant will precipitate all proteins, and some proteins will be difficult to resuspend following precipitation. This is especially a problem when a picture of the total protein content is desired.
A combination of TCA and acetone is the most common precipitant in 2-D PAGE studies, as it is more effective than either of these reagents alone. Besides, very few proteases are active in 10% TCA. Resolubilization is easier after precipitation with acetone alone (75% final concentration), but this gives a less complete precipitation.
A. Add 10% TCA in ice-cold acetone with 20 mM DTE to the sample.
B. Leave at –20°C for 2 h.
C. Centrifuge at 10,000g for 10 min.
D. Wash with cold acetone containing 20 mM DTE.
E. Repeat wash.
F. Let the pellet dry to remove residual acetone.
G. Resuspend pellet in lysis buffer.
2.2 DNase/RNase Treatment
If nucleic acids are present in high amounts, the sample will appear viscous and a smear will be seen after silver staining. If ultracentrifugation does not solve the problem, enzymatic digestion will.
A. Add 1/10 of the sample volume of a solution containing 1 mg/mL DNase I, 0.25 mg/Ml RNase A, and 50 mM MgCl2.
B. Incubate on ice for 20 min.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
