Yellow Western Experts!
My last blot came out looking like this wonderful picture.
It is absolutely maddening, I dont know whats causing this crazy background. I do the exact same thing and sometimes it comes out like this and sometimes it comes out good. What is the problem?!?
All solutions made fresh, filtered, etc
I should mention that this time after I opened the cassette, I noticed there was a slight tear on the filter paper on the side of the gel.
I was thinking if thats what caused the massive dark spot in the center, however, there is high background throughout. Then again, who knows?.
I keep seeing this dark spot and I cant figure it out whats causing it
It reappers with different primaries I use. All primaries are monoclonal
All primaries are are 1:1000 and 1:15000 for secondaries
This blot was 1:20000 for the secondary
The good images where 1:15000
same detection and exposure
My main concern is that I keep seeing this spot in the middle with different antibodies. I am trying to figure out what is causing this.
The blocking solution is 4% BSA
Im going to strip and reprobe this for actin tomorrow. I practically dont ever get background with actin. So if I dont get the background with actin, that seems to be the antibody saturation problem?!
Also, if you reprobe with ECL on PVDF is it suffice to wash the membrane for 5-10min and reapply ECL
Many thanks!!
I try to cram to many things, that why it comes out so long
Did your blot fall down or touch something else before you blocked it? How long did you block the blot? Are you using PBST or TBST? try using the latter. What is the concentration of tween that you use. If it's 0.1% try again with 0.2% Tween with a fresh gel (including blocking). How long did you wash the primary and secondary, try increasing the time and speed of wash if problem persists.
If you get the same problem with another primary, then most probably the blot touched something prior to blocking, and if nothing touched the blot it is a blocking issue. If another antibody gives clean blot, check your primary dilution, and see if using lower dilution helps.
how do you add the ECL to the membrane? did you check whether is evenly distributed on the membrane?
when you said you tried different Abs, did you try changing both primary and secondary? have you tried blocking in 5% milk in TBST?
have you tried using a different cassette when you expose your film? have you tried different films (although I don't think the latter is the problem is always good to check)? is this the only Ab you have this problem with?
I would say the high background is because of nonspecific binding of antibody and blocking buffers can reduce this background.
Try blocking in 5% milk in TBST. Use fresh TBST solution.
Nitrocellulose membrane is better than PVDF.
Wash the blot for three times 10 min each after blocking and antibody incubation.
All the best.
I think also that the blocking is the problem. Try 5% dry milk in TBST over night at 4 degrees C. And wash the blot after the primary antibody even 5 times in TBST, 10 min. each. Try nitrocellulose membrane for a heavy protein. PVDF membrane is better for the light ones, it is more sensitive.
Good luck
I do not think that blocking is the problem since the ECL is not lighting up all over the membrane, but mostly in the middle. I would ask how are you putting on your primary, and secondary - is it in a tray on a shaker or in a tube on a rocker? Also how are you adding the ECL? I found when I started doing westerns that squishing out the ECL is really important and you need to be very thourough!
Good luck
Andy
I did sometimes that kind of blot. Watch carefully whether your blot moves freely during washing. Increase the amount of wash buffer (this is easy). Sometimes the blot has some distortion and a lot of buffer needs to move freely. Or simply it just sticks to the dish. Lift a bit to get moving.
try to increase the blocking time, and before developing, u need to wash the membrane longer time: 3 times, each time for 10 min
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