enhancing western blot signals - incubation alternatives (Apr/14/2005 )
I'm so happy to have found a forum like this one!
I'm trying to figure out how to increase my western blot signal by using alternatives to the usual milk or bsa for incubation, but I can't seem to find anything online except for kits. Does anyone have any ideas or links to something I can make myself? My boss won't let me keep buying kits- too expensive- but I've had some pretty good results so I'm going to keep trying to figure it out!
It seems like the kit I've used somehow increases the ability for the primary to bind the protein, as the recommended secondary dilution is 1/20000 and the signal is very good, vs the almost blank blot I get using milk or bsa with same primary dilution (1/100) and various secondary dilutions from 1/1000-1/20000.
As I figure this out I'll continue to post so we can all get better blots from uncooperative westerns!
Thanks in advance,
The use of a good blocking agent would help reduce the background and thus you would have a better signal to noise ratio, therefore enhancing your western blot signal. Personaly, I have used 5% skim milk with great success, but if you are detecting a phosphorylated form of a protein using anti-phospho Thr/Ser/Tyr antibody BSA would be a better choice for blocking. However, another important factor envolved in enhancing the the signal on your Western blot is labeling your primary antibody directly or the selection of an appropriate substrate. For example utilizing a chemiluminscent substrate with your secondery antibody will enhance your signal significantly as compared to clormetric substrates. To enhance your signal further combine that with biotin labeling of your primary antibody or using a biotin labeled secondery antibody with Strepataviding-HRP.
Thanks for the tips!
To clarify: I have been using a product that enhances western blot signals five or more times. I don't know how it works, other than I have done a few more tests, it appears the solution that the primary is being icubated with actually provides better binding conditions for the antibody to bind to the epitope. There is no enhancement of signal by a tagging system such as ABC ( avidin/biotin) , only better binding conditions for the primary antibody / bound protein interaction. In other words, this mystery buffer gives five or more times better signal than when I use 5% powdered milk by providing optimized binding conditions for the primary.
What could this buffer be?
Does anyone use anything besides 5% milk/TBST to incubate their primaries with?
Is th product from Pierce, if yes which one is it Qentix or Miser?
I am quite familiar with these products and interested to know how effecient these products in enhancing the signal with use with different antibodies and proteins for WB, as it has been claimed to enhance the WB signal up to 10 times with most antibodies. Although these products are prepriotery, I think the treatrment of the membrane with either product will have an effect on increasing the surface area of the blot where the proteins are bound and thus exposing more sites for the protein (antigen) and thus more antibody binding with lower antibody concentrations. I hope this information is of help to you, and hope to have more people sharing their experinces with these or similar products. Good Luck!