I wish to perform western blotting for ubiquitin. In my earlier attempt I could not see the band corresponding to 8.5 kda in a 12% SDS gel. Please help me.
Try gradient gel with 20% max
http://www.bio-rad.com/en-us/sku/456-1096-4-20-mini-protean-tgx-gel-15-well-15-ul
Normally in a 12% SDS-PAGE gel, the size of protein you are trying to visualize will be almost at the bottom. Try higher % gel (15 or 18%) to achieve the visualization.
In mammalian cells only a small percentage of ubiquitin is monomeric. Most of is found in polybuiquitin chains or attached to proteins, so monomeric form would be very minor component of all ubiquitin pool in a cell. Secondly, sensitivity of ubiquitin antibodies is increased by autoclaving the WB membrane (http://hamptonlab.ucsd.edu/Protocols/AutoclavingBlots.pdf)
A hand-made 15% PA gel is good enough if you expect only monomeric band at 8.5, but if you want to see a ladder of poly-ub chains in cell extracts, the gradient gel is certainly better.
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