Hi,
Recently one of my blots came out looking like this,
I am trying to figure out whats causing this problem, the concentration of the antibody or something else.
My blotting protocol is
3x washes in TBS (with Tween 0.03%) after transfer
block o/n in 4%BSA
3x washes in TBS
1 hour primary at RT
3x washes in TBS
1 hour secondary at RT
3x washes in TBS
apply ECL 1 min
Could anybody tell me what's causing this
This blot was reprobed using 0.1M glycine (pH 2.5)
Hi, Aleks, is it "primary" WB, or after reprobing with the other pronary antibody? It looks like that some problem was with an-specicif binding of the primary antibody. Are you sure about antibody specificicty?
Also, you can try instead 1 hour RT 4°C overnight incubation with primary Ab, and wizthout any shaking.
Good luck!
If it is a re-probed membrane, it seems that you had some issue with the stripping procedure. Try wetting the membrane with Methanol well and wash it well after stripping. Then, block for 1 hr.
Also, another very possible thing is that the specificity of the primary antibody. Try incubating the antibody with the membrane in 4 degrees overnight instead of 1 hr at RT.
Hi Aleks,
Such a strong signal after 1 min. means non specyfic binding as Taras, Christopher and Ahmad indicated. Just to simplify the story I would change into 5% milk TBST for all the buffers. I saw many times such an catastrophe with BSA, nicely resolved with milk;)
best - jan
Is your primary antibody diluted in the blocking solution?
It is not necessary to wash the membrane again after blocking. You can directly move to the incubation with the promary antibody.
You may also try a different blocking solution, e.g. 3% BSA 1% milk in TBST (preferably with non phospho-antibodies), or commercially available solutions. It may also be helpful to incubate the membrane overnight shaking at 4°C with the primary antibody.
Make sure you have an appropriate concentration of secondary antibody (not too high). Another idea would be to change the secondary antibody or ECL.
Good luck! Sandy
Many thanks everybody
I have used this protocol before with ECF as the detection reagent and it worked fine with the dilutions I am currently using.
The BSA solution is always made fresh and is filtered.
Yes the both the primary and the secondary solutions (separately) mixed into blocking solution
While I was reprobing this blot, I also was reprobing another blot for actin
exact same conditions, exact same blocking solution and the other blot is clean
I forgot to mention beforethe protocol I use for stripping the blot after the 1st detection
I do three incubations in 0.1 M glycine pH2.5
10seconds, 1 min and 10min with intermediate rinses with wash buffer
wash 3x 10min
then I block for 30min in 4%BSA
WASH 3x 10min
1hr primary at RT
3x washes
1 hour secondary at RT
3x washes
apply ECL 1 min
I recommend to block the membrane at least 1 hour at RT and then skip the additional washing step after blocking.
Why rinse before and after blocking? I don't find that necessary usually. Also, is your TBST filtered.
I's also recommend a change of blocking solution. We often have a lot of background with 5% BSA which looks nice with 1% milk (if 5% ist too strong).
I recommend try 5% milk to blocking and check the Ph the whashing solutions, also put Tween in whashing solutions.
I have had success with blocking with 3% BSA overnight at 4 degree to reduce background. I agree that it is not necessary to wash before and after the blocking. There is a good stripping procedure on Abcam's website if you think it is the stripping that is causing the problem; however, looking at the film that does not seem to be the problem. Try to block overnight, primary for 2 h at RT, wash 1 h with TBS, secondary 1.5 h at RT, wash with TBS 1 h and continue as you previously did.
I agree with Anberitha you don't need to wash before or after blocking. However, I find Tween to be quite diluted. I usually use PBS with tween 0.05% or higher, and washes are for 15 minutes each, no less. Otherwise my blot protocol is the same as yours, except for the BSA, cause we use 5% milk for blocking.
Some colleagues had issues with BSA before, so you might as well try blocking with 5% milk in PBS ;)
Hope you can achieve your results!
Best regards from Chile.
Aleks, this is an interesting piece of reading for you: It saved me a lot of time and effort (and TBS) in my westerns.
"An improved western blotting technique effectively reduces background."
http://www.ncbi.nlm.nih.gov/pubmed/12210190
If you ever use PVDF/Immobilon again, here is an excellent stripping protocol that's much cleaner than the glycine method.
"A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing"
http://www.sciencedirect.com/science/article/pii/S000326970900178X
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