Dear fellow researchers:
I've recently started performing WB, mostly with molecular weight markers to test the equipment and optimize I/V conditions. This time I also ran protein lysates from two different cell lines. The gel rans fine and the transfer too was OK. I stained the first gel with Beta actin which showed crisp bands (Image 1). However, the second gel, stained with MMP-9 showed a lot of artifacts (Image 2). I use 5G3 Ab (MMP-9) from Thermofisher which has recommended dil from 1:500 to 1:2000. I used 1:1000. All conditions between membrane 1 and 2 were identical, including secondary Abs since both primaries were Ms. However, membranes gave different results. I used Supersignal West Pico plus as ECL reagent. Can someone suggest what might be the reason for this?
Lane 1-4 and 7-10 and Mol. Wt. markers.
Lane 5, 6 have proteins.
Hi Udesh,
to me it seems that your blotting conditions are good and it is just the MMP9 antibody that is not very good. You have very high background (that's why you see your ladders as well). How did you wash your membrane? With TBS-T? You can try harsher washing with SDS buffer (up to 0.5 M NaCl and 0.2% SDS in your TBS-T buffer). Try first short wash (2-5 min) and after you develop the membrane, you can wash longer to get rid of all the background. The rest is looking fine.
Good luck!
Katerina
Thanks for the input, Katerina. I’ve validated this MMP9 Ab for IF and it has shown very specific binding. This was the first time using this Ab for Wb. I washed the membranes using TBT-T. Are you suggesting that I wash the membrane after immersing them in ECL reagent?
In my experience, the antibody can be good in IF, but still it doesn't work well for WB. What I usually do: I first develop the blot normally (wash in TBST and develop with ECL). If the signal is not nice and has too much background, I follow with one TBST wash and a short wash in SDS buffer, followed by 2 washes in TBST and then I develop again with ECL. If the signal is not optimal, I prolong the SDS wash and develop the membrane again.. If you have more questions, don't hesitate to ask!
Your problems are because of the following reasons:
1. The primary antibody is not very good. Try using a monoclonal antibody such as rabbit monoclonal MMP9 antibody available from abcam.
2. You are using too high or too low concentrations of the antibody. Try to determine the appropriate concentration of the antibody.
3. Remember MMP9 is basically a secretory protein and it might be present only outside of the cells and it is also Zn++ dependent. So, may be you can do a correlative immunohistochemistry along with the western blot.
Regards
Hi Katerina Jerabkova I tried diluting the Ab a tad more than the last time and there was comparatively less noise. I also tried washing the membranes more robustly using TBST. I haven't used the wash buffer that you suggested. If the signal doesn't get better in the next run with more diluted Ab, then I'll seek to your method.
Nurul Kabir Thank you very much for the help. The Ab I'm using is mouse monoclonal which is supposed to show high specificity since it is monoclonal. It is likely that I was using a high concentration of the Ab. You may be right that since MMP-9 is a secretory protein so it may not show up on WB. Perhaps that's the reason why the protein did not show up on the WB. Thanks again for the information.
Yes, and that's why it is essential to do correlative immunohistochemistry. Even if the protein is secreted it should remain in the tissue and in a recognizable conformation because of the presence of zinc++.
Regards
Nurul Kabir I've tested it for IF and I can locate the protein intracellular which means that protein is present in the cells. Ideally, during the lysis step of WB, the protein shall be present in the cell lysate which makes me believe that the protein is present; I. just need to optimize the conditions for WB.
There are various sources of uncertainties. Maybe, blocking was done indequately. Another option is the antibody used, which is maybe not specific (for ref. see Baker M. Reproducibility crisis Blame it on the antibodies. Nature. 2015_521.p274-6. doi 10.1038521274a). In general, I would propose to check and (possbly) revise the protocol of the done experiments and analyses.
Andreas Eisenreich I had run beta Actin parallel with identical conditions which makes me believe that blocking may not be a problem or it should have been a problem in beta actin also. You may be right that the Ab isn't good for WB. Thanks for the reference.
Hi Udesh
In line with Katerina's recommendations, I think the presence of those artifacts it's likely caused by unspecific binding of MMP-9 Ab. In my experience, this can be solved by diluting the primary Ab, sometimes even further than the recommended dilution (i.e 1:2500 - 1:3000). You can even cut the membrane and incubate each lane with an increasing dilution of the Ab to determine which one gives you a better signal in the ECL.
This approach may diminish signal intensity, but increases Ab specificity and allow to see small differences between lanes more clearly.
If this doesn't work, you should check the blocking conditions or try with a more thorough washing.
Best of luck!
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