In my current experiment I'm trying to detect cytokeratin 10 by western blot in whole cell lysate of A431 cells. Unfortunately there are strong signals from non-specific binding in my blots (see attached pics).
On the attached Image1 left membrane probed with anti-cytokeratin 10 antibodies 1:200 and secondary HRP-conjugated anti-mouse IgG. Membrane on the right probed with different primary IgG and same secondary antibodies. HRP substrate - SuperSignal West Femto. Blocking solution - 5% Milk in TBST. Gel - NuPAGE 4-12%, running buffer - NuPAGE MOPS. Procedure for both membranes was exactly the same, except primary antibodies.
I tried to reduce non-specific binding by increasing primary antibody dilution. Image 2 - same procedure as for Image 1, but dilution of primary antibodies 1:2000.
Please advice what can be cause of such a strong non-specific binding and how can I avoid it?
Hi Oleg,
You could try the assay blocking the nitrocellulose sheets in 3% Nonidet P-40 (nonionic, non-denaturing detergent) in PBS, for 30 minutes. Followed by three washes with PBS before the incubation with the anti-cytokeratin 10 antibodies. Another thing that you could try change milk to fetal bovine serum (FBS) for your blocking solution.
Hi Oleg:
Your protocol looks all right if your protein loading amount is not so small. If your budget is not so tight, i would highly recommend you to buy another Ab from reliable company, such as that from Abcam, Sigma, which normally validated by the Abs and showed their results on the website.
Anyway, time is always more expensive than money.
Best regards
Shiqiu
hi
1- you should wash the PVDF more than 3 times for 10 min by shaking 60 rpm(PBS-T or TBS-T) .
2- please used from fresh and clean runing and transfer buffer.
3- wash pads with DDW before transferring .
4-primary antibody incubation 1:200 is good , its better increase incubation time (over night at 4c)
5- decrease secondary antibody concentration ( for example anti-mouse-HRP 1:5000 change to 1:10.000)
6-detrmaine cell lysate concentration by BCA kit
7- setup western blot by anti-beta actin
8- setup western blot for anti cyto10 used by diffrent amount of lysate (such as 10ug/well or 20/30/40 ug)
good luck
Thanks to all of you. I'll consider your suggestions and come back with some results later on.
Hi Oleg Dobrokhotov .
I used to get non-specific binding when I first started doing Western blotting. Consider the following:
- Use BSA as a blocking solution instead of milk.
- Increase primary Ab and decrease 2nd Abs.
- make sure you are using the right concentrations of the running and transfer buffers.
- Increase Ab exposure (time) to get the image.
Hope this helps
Once again, thank you for you suggestions.
A short conclusion from my investigation, maybe could be helpful for someone:
1) Most likely root cause for high non-specific binding - Ab degradation (see results of electrophoresis - EP.png; big fat band in the middle of the gel ~50 kDa is BSA from Ab buffer). Honestly, I didn't expect that they are so degraded, since I time to time use them for IF and they works as good as new...
2) I've also tried several combinations of blocking solutions and diluents for antibodies among available in the lab. Among them commercial blocking solution from Nacalai Tesque (Blocking One) works the best (see results of WB - WB.png)
Description of WB.png (from left to right; all solution in TBS-T):
1) Blocking - 5% Skimmed milk; diluent - 5% BSA; 1:500 primary; 1:10 000 secondary
2) Blocking - 5% Skimmed milk; diluent - 10% FBS; 1:500 primary; 1:10 000 secondary
3) Blocking - 10% FBS; diluent - 10% FBS; 1:500 primary; 1:10 000 secondary
4) Blocking - expired Amersham ECL Blocking Agent; diluent - 5% Skimmed milk; 1:200 primary; 1:10 000 secondary
5) Blocking - Nacalai Tesque Blocking One; diluent - 5% Skimmed milk; 1:2 000 primary; 1:10 000 secondary
For #4 and #5 I reused antibodies from previous experiment.
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