Hi all,
I am facing a problem in getting cleaner blot using an antibody against Polyubiquitin conjugates (FK1, Enzo life science) from an Immunoprecipitated sample. I have used both 5% Milk or BSA as blocking, tried different time points of blocking, along with different percentage and Buffers. It is not working well in any situation. I have tried two different dilutions (1:1000 and 1:1500), the problem persists in both. To develop the blot, I have tried least sensitive to high sensitive kits (Pico/Femto, Thermo). Increasing the washes is giving me fainter bands with similar background. Any suggestions?
Hi,
I can see that you have tried almost everything. Can you tell me what membrane you are using? Nitrocellulose or PVDF? I found that some antibodies work much better with one or the other, therefore it may be worth trying the nitrocellulose or PVDF depending which one you are using at the moment.
Best,
Maciej
I have tried both nitrocellulose and PVDF, though background was bit less in PVDF but it was not satisfactory.
Hi Maciej,
No, The sample is immunoprecipitated with different antibody and the efficiency of immunoprecipitation is really good. This antibody is to check the polyUbiquitinated conjugates of the immunoprecipitated protein.
No, The immunoprecipitated antibody is grown in Rabbit (IgG) while the PolyUbiquitin is mouse (IgM).
here it is. the best I got so far. 7% BSA blocking for three hours, 1:1000 antibody primary, 1:10,000 secondary. 5 washes for 10 minutes each.
Thanks for uploading the photos. From my experience the background looks like a dried membrane (this is particularly true for nitrocellulose, however, PVDF may also give such signal when dried). Can you explain how you applying the ECL reagent. Do you dry the membrane afterwards? I tend to keep the membrane wet in ECL reagent between two acetate sheets and expose to film while still wet. Maybe you can try it too?
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