GGS LIVE - Western Blotting

Method: Protein Electrphoresis and Western Blotting

About: Simply saying protein electrophoresis is a technique that allows to separate mixture of proteins by either their size or charge. After protein separation specific antibodies are used to detect and analyse protein of interest (western blotting). Before you go further please have a look at this animation about the

protein electrphoresis Biosolutions Blog - Protein Electrophoresis.

What: Detection of protein A, B and C in human protein extracts.

How: SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) is a form of protein electrophoresis where detergent (SDS - to see the structure click here SDS structure) is used to give all proteins a negative charge so they migrate in the same direction in the polyacrylamide gel. Proteins migration in the polyacrylamide gel is similar to DNA migration in the agarose gel. Concentration of the polyacrylamide (usually expressed as %) defines gel pores size. Small protein easily get through the pores and migrate faster where bigger proteins do not and migrate slower.

In this experiment we are going to analyse five different human cell lines (called clone A, B, C, D, E) for presence of protein A, B and C. Before we separate proteins by gel electrophoresis we need to prepare our protein extracts. There are different ways of protein extraction but most of them contain cell lysis, debri (cell membranes etc) and DNA separation and protein concentration quantification steps. Example of a such extraction is presented on the figure below:

After protein extraction and concentration quantification from Clones A to E we prepare samples for gel electrophoresis. We take the same amount of the protein for each extract. What I mean here is that for example we take 30microg of the proteins from each of the Clones A-E. Suplement that with the sample buffer which contains:
- reducing agent, usually beta-mercaptoethanol (to reduce disulfide bonds in proteins - denaturation),
- glycerol, which makes our samples heavier and easier to load on the gel,
- dye, usually bromophenol blue (makes our sample loading visible),
- dergent, a sodium dodecylsulfate (denaturates proteins).

After sample preparation, proteins are boiled (at 95 degrees Celsius for 3-5min) and loaded onto gel, like you can see in the figure below (remember that ladder, a protein reference mixture has to be loaded as well).

Dark blue bands which you see here represents proteins (in the real life, we would not see proteins separating) and light blue bands are ladder proteins. If we are using a prestained marker we would see ladder bands on the gel separating in real time:). After the electrophoresis, proteins are transfered to a membrane beacause working with gel in subsequent steps would be very hard and chellenging (polyacrylamide gels are very fragile). There are different types of membranes (for example PVDF - polivinyldiene fluoride, or nitrocellulose) and all of them have high affinity to proteins. In this step membrane is placed on the top of the gel and squeezed together in a special cassette. Again current is applied and proteins migrate from the gel and stick to the membrane (see figure below).

Notice that position of the proteins on the membrane is the same as on the gel before the transfer. Observe also that other binding sites on the membrane are still unoccupied. Because membrane has high affinity to proteins we need to block those sites. Blocking of this sites is necessary as in subsequent detection step we will use antibodies (which are also proteins) and those could stick to the membrane giving us confusing background signal (see figure below). Usually for blocking we use source of neutral protein like low fat milk, BSA (bovine serum albumin) or FBS (fetal bovine serum).

After blocking membrane can be cut into pieces and then each piece is incubated with the specific primary antibody which recognize the protein of interest (usually overnigth, at 4 degrees Celsius) and then with secondary antibody (1hr, at room temperature) that recognize the former one. Usually secondary antibody is conjugated to another molecule which is capable of producing signal that latter is detected. In this case it is a Horse Reddish Peroxidase (HRP) an enztme, which catalyse reaction and one of the products of that reaction is a photon. Complex Protein-Primary Antibody-Secondary Antibody (called a sandwich complex) is formed and a substrate of HRP is added. This way we can visualize the protein of interest (see figure below).

Membrane is placed in a special cassette and photographic film is placed on it. Film is exposed in the place where the protein of interest is present. Developing the exposed film reveal presence of the protein (see figure below).

After developing, proteins appear on the film as different size bands. As you remember in our experiment we have been analysing five different human cell lines (Clones A-E) for presence of the proteins A, B and C. Notice that for each experiment, membrenes were incubated with antibodies specific for protein of interest and control protein. The control blot is performed because it tells us that we have loaded the proteins onto gel and and additionally, it tells us if the amounts are equall between the samples.

Lets have a look on our results.

The membrane on the left hand size show analysis of the protein A. Firstly look at the control protein. You can see that the amount loaded is equall everywhere. You can clearly see that protein A is present in all of the analysed cell lines and additionally that levels of protein A between Clones A-E is the same.

The middle blot show the same for the protein B (in this experiment we analysed only Clones A-D). In this case protein B is present in Clones A, B and C but not in the Clone D. We can be quite sure about that conclusion because we did loaded proteins as the control protein is present in that sample.

The right hand size membrane show analysis of protein C. And similarly you can see that proteins are loaded equally and that protein C is present in all of the samples analysed. On this blot we can additionally see that  protein C is being phosphorylated in vivo (what means within cell). Protein phosphorylation can be detected by Western Blotting because usually phospho forms of proteins migrate differentially than unphoshorylated forms. In this case phospho form of protein C migrates slower and appears as a shifted band above the unphosphoryalted form of the protein. Conclusion that can be draw from that blot is: protein C is present in the Clonses A-E and additionally that protein C exist as a phosphoform in the Clones C-E.

Remember that analysis of the blots must be performed only in the presence of appropriate controls.

That is it:)

I hope you like it:)

Maciek GGSTEAM