Anti-Sumo 1 antibody (ab11672)

no staining on western blot using prostate cancer cell lines PC3 and LnCaP cell line extracts, 50ug protein loaded per lane with primary diluted 1:1000

ANSWER:

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Hi! I have tested using different blocking agents such as BSA and serum but it didn't help. Have you got a recommended protocol? Best regards,

ANSWER:

We forwarded your protocol to the source of the antibody and we have just received this information:

" I do agree that the anti-Sumo antibody it present a basal background in the western blot, but at least in the conditions used in the lab, we always have been able to see the Sumo band. However, due to the enquiry, I performed new conditions to try to improve the signal to background ratio.

The conditions used for this western blot are similar to the conditions used by the user but changing several things:

1- dilution of the primary: 1:3000 2- in all the incubations TBST-0.7 (Tween: 0.7% !!!, yes, 0.7%) was used.

Other details that might be important:

3- incubation time for the antibody: 1h 30 min at RT 4- membrane used for the transference: nitrocellulose (Scheleicher & Schuell, Protran BA 85, pore size: 0.45 um) 5- might be important the amount of extract added: in the figure it was loaded around 50 ug of total cell extracts 6- we used west pico luminol products by pierce... very sensitive ECL might give a strong background. (the rest it is pretty much the same as described by the user: primary and secondary diluted in TBST-0.7 + 5% skimmed milk)."

I hope this helps you, if you still have problems please get in touch with us,

BATCH NUMBER 56867 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM I bought the SUMO 1 antibody and a SUMO 2+3 antibody two weeks ago. The SUMO 2+3 antibody works perfect but the SUMO 1 antibody only gives a very high background, the membrane is totally black after just 1 sec of development. I have used 5% non.fat dry milk as blocking reagent for both the SUMO 1 and SUMO 2+3, have tried TBS and PBS (with o,1% tween) and have used a anti-rabbit HRP linked secondary antibody for both (same membrane as well). It seems like the primary antibody binds to the whole membrane, what is wrong? The antibody? Have also tried the SUMO 1 antibody in just TBS and PBS, but also with blocking buffer but havent got any better results

SAMPLE Cell extracts that has been affinity purified in two steps. The eluate work perfect with SUMO 2+3

PRIMARY ANTIBODY SUMO 1 ab 11672 SUMO 2+3 ab3742 1:1000 diluted, 4 degrees over night

SECONDARY ANTIBODY AntiRabbit HRP-linked, Cell signalling Technologies room temperatute, 1 hour

DETECTION METHOD Supersignalling, PIERCE

ANTIBODY STORAGE CONDITIONS 4 degrees

ELECTROPHORESIS/GEL CONDITIONS 4-12% BisTris gel NuPAGE, both MOPS and MES hev been used

TRANSFER AND BLOCKING CONDITIONS TGM buffer, Semi dry blot (BioRad), 45min 15V. 5% w/v non-fat dry milk

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 8 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

I'm very sorry for the delay in replying to you, we have been trying to get in touch with the academic who has provided us with ab11672 and unfortunately it has proven very difficult. We are still trying to find out a recommended protocol, my sincere apologies for the delay.

I would like to ask if you have tried a different blocking agent, like BSA 5% or serum? If after testing those you still have the same problem I will of course send you a replacement antibody.

I look forward to hearing from you, thank you for your patience,