Electrophoresis is the motion of iyonized molecules under the electric field. Nowadays for protein separation, poliacrylamid gel electrophoresis (PAGE) is used. Generallay electrophoresis is used to separate molecules according to their molecular weight. This situation is also same for poliacrylamid gel electrophoresis but since proteins can have very large physicochemical properties, it would be more effective to separate proteins by based on additional properties such as isoelectric point differences. There are two type of protein separation by based on electrophoresis that are 1-D PAGE and 2-D PAGE.
In this separation process, proteins are separated according to their molecular weight. In the Western Blot method, protein sample are loaded into the well and if their amount is enough for visualization; after antibody incubation, interested protein can be determined.
In our unit, visualization is performed by chemiluminescence assay. The logic behind this assay is the use of Horse Radish Peroxidase enzyme which is tagged with secondary antibody. The required antibody must be supplied by researcher. In addition to interested protein antibody, reseacher also need to supply an antibody for the housekeeping protein which is required for quantification of results. (3)
Generalized steps for the Western Blot;
– First step is the protein loading into the well. The amount of protein can be changed according to the study but generally 40 ug protein is used.
– After that semi-dry or dry blotting is performed and the proteins are transformed to PVDF membrane.
– After blocking step, proteins are incubated with primary and secondary antibodies.
– As the last step chemiluminescence visualization is performed.
In the two dimensional polyacrylamide gel electrophoresis, protein mixture are separated initially according to their isoelectric point (pI), and then to their molecular mass.
Before separation steps, the proteins are loaded into the strips which have different pH gradients. For this purpose the strip gel is rehydrated with a buffer and protein. Therefore at this rehydration steps, proteins are loaded into thin strips. Strips are incubated with buffer and protein in BIORAD PROTEAN IEF Cell for 12-16 hours.
After this steps the strips are placed BIORAD PROTEAN IEF Cell and for the first separation step, protein mixture is placed onto strip and run on BIORAD PROTEAN IEF Cell for their separation according to their pI values. Then strips must be equilibrated to denaturate the sample proteins by using reducing agents like DTT. After DTT treatment, proteins are incubated with IAA for alkylation purpose.
Lastly, they are placed onto the PAGE and run in the BIORAD SDS-PAGE tank.
After this second separation step, the gel photo is taken with Bio-Rad’s Proteome Works VersaDoc Gel Image Acquisition system where the protein spots can be analyzed. The analysis of protein spots are performed with PDQuest Gel Analysis System. To characterize the content of the spot of interest, the spot is excised from the gel by Bio-Rad’s Proteome Works Plus Spot Cutting System.