Sodium orthovanadate preparation
All steps to be performed in a fume hood.
a. Prepare a 100 mM solution in double distilled water.
b. Set pH to 9.0 with HCl.
c. Boil until colorless. Minimize volume change due to evaporation by covering loosely.
d. Cool to room temperature.
e. Set pH to 9.0 again.
f. Boil again until colorless.
g. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling.
h. Bring up to the initial volume with water.
i. Store in aliquots at - 20°C. Discard if samples turn yellow
3. Preparation of lysate from cell culture
(1) Place the cell culture dish in ice and wash the cells with ice-cold PBS.
(2) Drain the PBS, then add ice-cold lysis buffer (1 ml per 107 cells/100mm dish/3750px2 flask; 0.5ml per 5x106 cells/60mm dish/1875px2 flask).
(3) Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microfuge tube.
(4) Maintain constant agitation for 30 minutes at 4°C.
(5) Centrifuge in a microcentrifuge at 4°C.
You may have to vary the centrifugation force and time depending on the cell type; a guideline is 20 minutes at 12,000 rpm but this must be determined by the end-user (e.g. leukocytes need a very light centrifugation).
(6) Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.
4. Preparation of lysate from tissues
(1) Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.
(2) Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to “snap freeze”. Store samples at -80°C for later use or keep on ice for immediate homogenization.
For a ~5 mg piece of tissue, add ~300 μl lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 μl lysis buffer, then maintain constant agitation for 2 hours at 4°C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml).
(3) Centrifuge for 20 min at 12000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.
The buffer (with inhibitors) should be ice-cold prior to homogenization.
5. Determination of protein concentration
Perform a Bradford assay, a Lowry assay or a BCA assay. Bovine serum albumin (BSA) is a frequently-used protein standard.
Once you have determined the concentration of each sample, you can freeze them at -20°C or -80°C for later use or prepare for immunoprecipitation or for loading onto a gel.
6. Preparation of samples for loading into gels: denatured and native, reduced and non-reduced.
(1) Denatured, reduced samples
Antibodies typically recognize a small portion of the protein of interest (referred to as the epitope) and this domain may reside within the 3D conformation of the protein. To enable access of the antibody to this portion it is necessary to unfold the protein, i.e. denature it.
To denature, use a loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95-100°C for 5 minutes. Heating at 70°C for 5-10 minutes is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently.
The standard loading buffer is called 2X Laemmli buffer, first described in Nature, 1970 Aug 15;227(5259):680-5. It can also be made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in a 1:1 ratio with the sample.
Laemmli 2X buffer:
4% SDS
10% 2-mercaptoehtanol
20% glycerol
0.004% bromophenol blue
0.125 M Tris HCl
Check the pH and bring it to pH 6.8.
When SDS is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. SDS denatures proteins by “wrapping around” the polypeptide backbone. SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - i.e., the denatured polypeptides become “rods” of negative charge clouds with equal charge or charge densities per unit length.
In denaturing SDS-PAGE separations, therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. SDS grade is of utmost importance: a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS.
It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size by using ?-mercaptoethanol or dithiothreitol (DTT).
Glycerol is added to the loading buffer to increase the density of the sample to be loaded and hence maintain the sample at the bottom of the well, restricting overflow and uneven gel loading.
To enable visualization of the migration of proteins it is common to include in the loading buffer a small anionic dye molecule (e.g., bromophenol blue). Since the dye is anionic and small, it will migrate the fastest of any component in the mixture to be separated and provide a migration front to monitor the separation progress.
During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution.
(2) Native and non-reduced samples
Alternatively, an antibody may recognize an epitope made up of non-contiguous amino acids. Although the amino acids of the epitope are separated from one another in the primary sequence, they are close to each other in the folded three-dimensional structure of the protein, and the antibody will only recognize the epitope as it exists on the the surface of the folded structure.
It is imperative in these circumstances to run a Western Blot in non-denaturing conditions, and this will be noted on the datasheet in the applications section. In general, a non-denaturing condition simply means leaving SDS out of the sample and migration buffers and not heating the samples.
Certain antibodies only recognize protein in its non-reduced form i.e. in an oxidized form (particularly on cysteine residues) and the reducing agents ?-mercaptoethanol and DTT must be left out of the loading buffer and migration buffer (non reducing conditions).
Protein State | Gel condition | Loading buffer | Migration buffer |
Reduced - Denatured | Reducing & Denaturing | With ?-mercaptoethanol or DTT and SDS | With SDS |
Reduced - Native | Reducing & Non- Denaturing | With ?-mercaptoethanol or DTT, no SDS | No SDS |
Oxidized - Denatured | Non-Reducing & Denaturing | No ?-mercaptoethanol or DTT, with SDS | With SDS |
Oxidized - Native | Non-reducing & Native | No ?-mercaptoethanol or DTT, no SDS | No SDS |
Rule of thumb: Reduce and denature unless the datasheet specifies otherwise.
