Hi,
I have trouble detecting a protein in a western blot. In my current blots I can’t see any bands at all.
In my experiments I am looking at different proteins in unphosphorylated and phosphorylated forms, using the unphosphorylated protein as a standard. For one of the proteins, I had successfully used the same membrane to detect other proteins before. I was even able to detect the phosphorylated version of the protein. I have tried the antibody in other blots with the exact same conditions before and had no problems.
I am even more confused now, because the antibody I am using is the same hostspecies as one of the other proteins I detected earlier on the same membrane, but now I can’t even see any of the old bands.
The same problem occurred in three different blots on the same day, using different antibodies, but the same buffer, membranes and ECL as before and after.
Does anyone have any suggestions regarding this problem?
There are many things that could have gone wrong.
One possibility is that the ECL has been activated by someone contaminating bottle B, with some of the solution from bottle A. That would lead to the activation of the reagent in the bottle and so it wouldn't matter when you mixed the two together prior to exposing. So that is where I would start with - change the ECL.
I don't know what molecular weight standards you use - but you can try using anti-penta his to see if some of the standards react to confirm that everything is working.
Dear Carolin,
If you don't detect your regular proteins anymore, try to prepare almost all solution afresh. May be there were some contaminaton in your PBST (TBST) or milk or something else. We had this problem when used PBS stored for 2 weeks in the fridge. And check your ECL too!
good luck!
If you are using a new batch of antibodies, you may also contact the manufacturer of the antibody. Sometimes you get a bad batch (it happened to me) and manufacturer are normally willing to help you out with a different batch.
Usually if you don't see bands which you saw before with same antibodies and conditions. The first thing, you've to check about ECL. We also had same problem recently, started changing everything including secondary antibodies yielded no clues. Once after we replaced ECL we are started seeing bands like as before. Later we found that our ECL was too old(Check date of expiry). our Western are fine now.
All The Best.
Thank you very much for your answers!
The confusing thing is that I tried a different blot with the same solutions (including tbst and ecl) and antibodies, but on a different membrane afterwards and it worked really well. I also detected the phosphorylated version of the protein on the same membrane, so I suppose that the membrane is fine and that the transfer worked.
So, you are confident that all your solutions were fresh and antibody worked properly...
Membrane contamination also can be a reason. Did you try to strip and re-block your membrane after previous detections?
I haven't tried stripping and reblocking yet. I didn't use to do it in previous blots either though, and never had any problems with the detection of other proteins on the same blot. Seems definitely worth a try in this case, though!
Hi Carolin,
your current issue could be Ab's related , samples' related or blot related.
I would suggest you to run your samples with a positive as well a negative control when you can, to understand if the mainly issue is due to the antibody or your sample only. After the blot you could use a Red Ponce solution to double check your transfer (don't worry it's a revertible staining: you can wash it away). If you didn't change the protocol for the proteins' isolation it should work. Bare in mind that some peculiar proteins needs a specific kind of membrane : if you were working on PVDF and the pore size accidentally changed (wrong order) also that aspect could affect your western. Currently it's hard to tell you more if you didn't run controls with your samples. I'm sure you will sort it out: just change one thing for time and you'll nail it!
I hope it helped somehow.
Cheers,
Micol
Along with all the suggestions given, you must also take into account that the nature of blocking solution used during western blotting also plays a crucial role in detection of phosphorylation and unphosphorylated proteins. Generally, one should use skimmed milk while detecting unphosphorylated protein whereas BSA works fine with phosphorylated proteins.
Here, you can try probing one antibody on the membrane, stripping the proteins and re-probing the same membrane again but using the different blocking solution. If all other reagents are working fine, the blocking solution might be interfering with the phosphorylation sites and thus preventing generation of any signal.
http://www.abcam.com/protocols/western-blot-troubleshooting-tips#No%20signal
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