hello everyone i have began my work on brms1 in mda231 and mcf7 i do western blotting to get brms1 endogenous but i keep getting nothing on my membranes except background bands i also tested the primary and secondary antibody and they are working fine any recommendation .
thank you
Hello Reham. Have you checked w Ponceau S staining to see a successful transfer on the membrane? Do you have a positive expression on your loading control (actin, GAPDH)? Is it okay if you share a picture of the membrane, the one with background staining? We can advise better if we can see the membrane.
hello nurul thank you for the respond , i will attach the pic here so you can see it . the one on the right its with 1:500 and the left on 1:200 of primary rabbit anti brms1
In my opinion, you have a successful transfer, as you can see faint bands. However, I think you have a low protein content. How much total protein did you load per well? I would go for 20-40 ug/well. But since you are working With mcf7, I can understand that you have a very slow growth rate. My tips on this will be to use a very minimal protein lysis solution when you extract them. I used only 50 ul of protein lysis per well of a 6-wellplate, so that the protein is concentrated.
Thank you i do another round of WB but i have a weird thing i think its ecl reagent i will upload the pic as soon as possible
Good job on the second membrane - successful transfer and higher protein content per well. However, now the coverage is inconsistent. You are right, it is maybe the ECL - potential reasons: too old solution, not covering all areas, incubation time is too short or too long. Or it can be that the primary antibody is not specific to your cells. Can we see your loading control (actin or GAPDH)?
hello Dr. Nurul thank you for helping me this is the last trial to see if primary and secondary antibodies work so first i load the proteins then block it with milk 5% then wash for 14 min then add primary antibody (1:200)that is 70ul in 14ml tbs / wash for 14 min then secondary antibody(1:200)that is 70 ul in 14ml tbs /wash for 14 min then incubate for 30sec in ecl 500ul from each reagent then read in i bright CL1000
Oh dear. This time, I am not sure the transfer is successful. However, is this an optimised protocol? The black background can be due to these: Dried membrane, Underwash, Concentrated antibodies, Bad ECL. Here is my protocol that usually works for me:
Block in 5% milk for 1 hour at 40RPM,
Wash w TBST 3x5mins at 40RPM,
1'Ab at 1:1000 (minimum) for 1hr RT at 40RPM,
Wash w TBST 3x5mins at 40RPM,
2'Ab at 1:5000 (minimum) for 1hr RT at 40RPM,
Wash w TBST 3x5mins at 40RPM,
ECL for 4.45mins.
Never touch the membrane. Use forceps all the time. Never stack up the membrane, Use clean containers.
Thak you Dr. Nurul iam really greatful i found out that the secondary antibody was the problem so i change it today i will do another round and update you with the result thank you so much
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