I have been working with western blots and got great bands for actin. After that I stripped membrane with harsh stripping buffer but then blocked for 3hours (5% non-fat milk). I was not able to see the proteins. I used ponceau stain and still proteins do not appear. What could be the reason for this.
Can I get suggestion on how to get rid of excess blocking. I was thinkjng of washing membrane in tbst for long time in room temperature.
Regards,
Pratik
hi
I do not think over blocking happened, but stripping Has been strong. if target protein at low level expression , its very difficult to get the band .check your paper with strong ECL substrate (for example Luminata Forte )finally you can check your paper with anti beta actin (at half concentration )again , if You did not receive signal or band , you must run the gel again .
good luck
Sometimes harsh striping buffer removes protein as well. Well it is har. One solution is there, wash with tbs. 30 min block with 5% milk. Add primary antibody for overnight with fresh one at 4 degree. Chances are you will get bands but not very clear. Try this.
It's up to your protein expression level. For those of high level, you can see good blotting bands even without re-blocking by TBST. For low level or sensitive proteins, I suggest you run new gel again. if precious samples, try to cut your membrane to separate blotting for proteins with big different molecular weight. Using striping buffer is the last choice.
Stripping always removes some protein and not only bound antibodies, therefore start with low abundant proteins first if they are on the same molecular level, commonly also phospho-signals first, then high abundant proteins. Cutting of the membrane is also a good idea if you have several proteins with a different molecular size. Use of TBST after stripping only ensures to get the right pH as stripping buffer is at a pH of approx. 2.2, where any milk or BSA will precipitate.
Thank you guys. I figired it out that it was old antibodies that were causing the lack of bands. I used fresh antibodies for same protein and got excellent bands.
Thanks once again.
Regards,
Pratik
Well, after a lot of problems I just omitted the whole non-sense of 'non-specific blocking'. The results were wonderful, very clean with no background.
I never understood this 'non-specific binding'. How can be the binding of antibodies which have one of the most specific binding of any reaction (the Kd is something like 10 to the power 15) be 'non-specific'? This misunderstanding appeared because of antibodies which are either impure or not good quality.
Get some good primary and secondary antibodies and all your problems with 'non-specific binding' will simply disappear; no BSA or goat serum!
At least try a few experiment with your present antibodies without any blocking steps. Try to spin down the final antibody solutions, do the incubation at 42 degree centigrade (and not 4 degrees) for 1 hr only. Hopefully, you will never need the blocking step.
Regards
To avoid losing protein in the membrane do stripping with TBST with a long duration of time and multiple washes in a shaker. This work well for me.
Better you cut the membrane in two half one for your protein and one for actin, and perform the blocking respectively and separately.
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