Dear All,
In my western blot experiment, I have problem with blotchy blackground (like in attachecd picture) on the blot, which usaully appear when I expose low signal band for 15-30 min or more by CCD camera with Amersham ECL Prime.
I read on Biorad's website for the causes, which is either dry or insuffcient wash. I don't think it is likely in my case. I always check PVDF membrane for mark before using; never leave membrane dry; cover with clean plastic before exposure; wash 4-6 time with TBST after easch antibody incubation.
How should solve this problem?
Thank you
Cheerio,
What primary/secondary antibody combination did you use? I noticed that anti-goat secondary AB tend to leave a lot of non-specific binding marks on membranes.
It also might be incufficient blocking after blotting. What do you block your membrane with?
Sometimes longer blocking time might help. You may also screen for the best blocking agent (BSA, milk, casein). Including Tween may also help, but check the best conditions for you experimentally. Finally, you may also consider screening for the best dilution of the secondary Ab.
Two recommendations:
Thank you all
Dear Dmitry, I used 5% non-fat milk in TBST as blocking buffer.. My protocol after transfer was: blocking membrane for 1h at room temp, wash briefly 2 time in TBST, incubate primary antibody overnight at 4 degree celsius, wash 5 min 4 time in TBST, incubate secondary 1h at room temp, wash 5 min 4 time in TBST, wash 5 min in TBS, remove excessive TBS, incubate with ECL for 5min, remove excessive ECL, and finally expose by CCD camera.
Don't wash off the blocking buffer. Block for 1 hour, immediately exchange buffer for primary antibody in diluent containing blocking components.
Wash for 10 minutes each wash. When it comes to WBs, longer washing will 99% of the time result in a cleaner blot. I've washed for hours on particularly messy antibodies to get a high quality final result.
Seems to be result of localized accumulation of antibodies/ECL reagents. As suggested by others, increase in blocking, washing and removal of ECL reagent should help. Make sure the membrane used is not very old (5 - 6 years old, kept in drawer with pack open) and always wear gloves while handling it.
Thank you All. I have rechecked it. The cause seems to be ECL. The blotchy or darker background are from the area that has more volume of ECL or has ECL longer on top of the membrane. I usually just pour drops of ECL to cover all membrane and just leave it for 5min. Should I shaker or how to pour ecl properly?
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