Quantitative Western Blotting to Estimate Protein Expression Levels
Transcript
Quantitative western blotting — a total protein normalization-based technique — is useful for quantifying target protein expression levels.
To begin, load tissue lysate into the wells of a polyacrylamide gradient gel. The lysate contains the target protein, along with other cellular proteins.
Run the gel at an appropriate voltage for the electrophoretic separation of the sample proteins along the gradient. Upon completion, place the gel on top of the polymeric membrane of a transfer stack.
Apply an electrical current to transfer the separated proteins onto the membrane. Next, place the membrane in a fluorescent total protein stain. The charged dye molecules in the stain bind electrostatically to all the proteins on the membrane.
Add a blocking solution. The proteins in the solution bind to the membrane, thus. preventing the non-specific binding of detection reagents. Add a primary antibody binding only to the target protein. Then, add a secondary antibody — labeled with a fluorophore — that attaches to the primary antibody.
Excite the sample with a laser. Record the emitted fluorescence from the dye molecules of the total protein stain and the antibody-bound fluorophores at their corresponding wavelengths.
To calculate the normalized protein loading value, divide the total protein signal of each lane by the highest value. Divide the target protein signal by the corresponding normalized loading value to determine the relative expression of the target protein.
