1. Maximize the number of antibodies you can test on one blot

Western Blot cut in stripsWhen testing multiple antibodies using the same lysate, try a gel with a stacking layer that has one large trough instead of one that has multiple lanes. Once the gel has been transferred to a blot, you can use a razor blade and ruler to cut the blot into strips of equal widths. In most cases, twice as many strips can be cut from one large trough than from cutting apart a blot with multiple lanes. The strips can then be incubated in a tray with narrow wells.

2. Visualize the proteins on your blots

After SDS-PAGE transfer, stain your blot with Ponceau S to confirm a successful transfer and to determine the exact alignment of lanes and placement of the proteins on the gel. Ponceau S will not interfere with antibody labeling of the blot.

Ponceau S staining of Western blot

3. Try BSA for blocking when using phospho-tyrosine antibodies

When working with phospho-tyrosine antibodies, try blocking and incubating with 3% and 1% BSA respectively instead of milk. Milk often contains high concentration of phospho-tyrosine and thus can give an abnormally high background if used as a blocking agent with phospho-tyrosine antibodies.

4. A protein’s molecular weight will affect its transfer time

Proteins of different molecular weights transfer from gel to membrane at different speeds and efficiencies. Larger molecular weight proteins take longer to transfer. Try staining your gel with coomassie after the transfer to confirm that the proteins of interest have migrated out of the gel. Remember to also consider protein loss across the membrane. By adding a second membrane behind the first, you can see to what extent the proteins have blown through. Process both membranes with your antibody. Use data from such experiments to optimize transfer time for the specific protein of interest.

5. Avoid freeze-thaw cycles

Antibodies are sensitive to freeze / thaw cycles and bacterial infection. If you don’t plan on using all of the antibody right away, freeze it in aliquots and/or keep it at 0-4 degrees Celsius with antibacterial agents such as bacitracin or azide.