Buffer and Western paper
The basis of protein separation in western blotting is the size of the proteins. Proteins in the presence of SDS and denaturants completely separate from each other.
Note: Acrylamide is a potent accumulated neurotoxin. Wear gloves when working with it.
Sample buffer (Loading Buffer)
- To load the sample, we use the loading buffer in an electrophoresis gel.
- The loading buffer contains bromophenol blue with a negative charge, that helps to samples move in the gel from the cathode to the anode (only based on the protein size)
- In this buffer, the blue color indicates the movement of the samples in the gel.
- The loading buffer used for each sample is proportional to the sample that has the highest protein concentration.
- The ratio of loading buffer to the sample is usually 1 to 1, since our samples may not fill the total volume of the well, and the samples begin to move with different volumes in the gel (this causes the samples to be raised and lowered in the gel), Therefore, the volume of all samples should be uniformly distilled using distilled water.
Paper (pvdf) or (polyvinylidene difluoride) and nitrocellulose (nitrocellulose)
- Both PVDF and nitrocellulose papers are used for western blotting andamino acid analysis. In general, PVDF paper has a binding capacity of highermolecular weight proteins and is more sensitive to nitrocellulose paper. Thisfeature of the PVDF paper allows for identifying less-expressed proteins.
- If you use this paper, there may be noise in your antibody detection step. On the other hand, nitrocellulose has a lower diagnostic sensitivity than PVDF, but it produces less noise when detecting bands. Typically, nitrocellulose paper is used to identify proteins with lower molecular weight.
Important points :
- A blocking buffer used for the western can be made either from powderBSA, (Bovine Serum Albumin) or from Skim Milk.
- Paper is incubated in a blocking buffer solution for two h at roomtemperature, or overnight at a temperature of 4 ° C.
- The paper is incubated in the initial antibody for two h at roomtemperature or overnight at 4 ° C . (although it should be noted that some ofthe antibodies rapidly degrade at room temperature, thus incubation must beperformed overnight at 4 ° C)
- The secondary antibody used for Western Blot should be conjugated withHRP or AP.