I am interested in confirming whether a targeting peptide we have developed is successful in binding its target receptor protein. To do this, I have been running pull down assays to detect expression of the surface receptor.
I have been running two western blots in parallel. In the positive control pull down, I am using AG labelled magnetic beads, incubated for 1hr with a monoclonal specific antibody, followed by 1hr with the cell lysate. This western blot appears fine.
In the second, I am using streptavidin labelled beads, incubated with the targeting peptide for 1hr followed by the cell lysate. This blot came out quite dirty, and was as though in a negative print; the background was dark, and samples appeared white.
I washed off the ECL incase it had over developed and re-imaged - at this point, the samples did not show up, although the ECL and colorimetric ladders still appeared fine.
I just wondered whether anyone else had had this problem, and what the reason could be?
Thanks very much for any advice!
Brittany
At the moment, yes. I use 2% BSA for phosphorylated proteins, but 5% milk otherwise.
if I understand well..you observe a white band where you expect a dark band...
better to post a picture..
but I think this is because the ECL reagent is rapidly destroyed , ie there is a large amount of your target prot, so use less protein sample, dilute your first and second ab
For streptavidin westerns, try using BSA for blocking as there is the off-chance that you're cross-reacting with extra biotin in your buffer.
Thanks for all the helpful answers! I will be re-probing this week with new antibodies, so I will use 5% BSA rather than milk and also load less protein. As I was trying to confirm pull down, I had been loading a high concentration which is a good point. Thanks again!
Brittany
Hi brittany. Most probably is the blocking solution or the amount of protein loaded. You should also check the photomultiplier (it should be not too high). But if that's not the case you should also check the quality of the salt in your incubating solutions (eg. TTBS or TPBS). You can also dilute the ECF with dH2O before use it in your blot.
I hope you can solve it.
Fernando
I have had this problem when my lane is massively overloaded. The ECL essentially burns out the center of the band when there is a ton of protein.
Thank you both!! Diluting the ECL is a good idea, i'll try that soon too. Overloading seems to be the most likely - as I was running a pull down for a receptor with low expression I was under-estimating the protein loading quite a bit. Thanks again! :)
Incase anyone else comes across this issue - it seems that it was a combination of two problems. First, I decreased the amount of protein loaded. (I had been loading high concentrations, as I was working will a streptavidin bead bound pull down of a protein with only low expression to begin with. However, the streptavidin dissociated from the bead kit was leading to the band excluding dyes. Problem one solved. The second issue appeared to be my blocking method. I changed from 1hr at room temp, to 1.5 in the fridge, and this resolved the issue of the remaining membrane appearing too dark.
Thanks for all your input!
Brittany
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