HELP! WESTERN BLOTS SUDDENLY STOPPED WORKING! - (Jun/17/2010 )

Hi guys,

I am looking at BDNF protein expression using western blots. I've done 20 or so of them in different regions of the mice brain and my westerns were all working fine (refer to picture: Working WB).

Recently i've been getting westerns that looked fine during ponceau stain but in the images, theres smearing clouds over my bands,messy-looking membrane and weak signals of my protein.

I have been doing everything the same way except the primary antibody has just been brought ( but same brand and using same dilution).

PLUS, my actins look fine so im thinking it must be my primary antibodies??

Please help! I've posted my membrane images.

Protocol i've been using after transfer:

- Blocking with 5% skim milk in TBST for an hour
-Incubate primary antibody (1:500) in 5% BSA TBST over night 4 degrees
-wash 15mins X 2
-Apply secondary antibody (1:2000) in 5% skim milk TBST for 1.5 hrs
-Wash 15mins x 3

I suggest

uneven shaking with inconsistent contact of membrane and solution especially blocking solution, or the membrane was concavely formed

I repeated the exp with different samples but same primary antibody. I made sure my shaking was consistent, membrane not concaved and that membranes were all covered by blocking solution...

I got the same results...really weak signals and cloudy/black patches everywhere albeit actin looked perfect...

I was wondering if it mattered that i mixed the last few micro-liters of my old primary antibody with my new batch of primary antibodies i just opened for these sets of experiments where i started to get these ugly smearing and weak signals???

Antibodies were exactly the same i.e. from same company, source (rabbit)..its the same product but ones old and ones a new batch we just purchased...

HELP PLEASE!

Inmost sun on Jun 17 2010, 10:20 PM said:

is the antibody titer the same as the old batch? is it the same lot#?

you may want to try different dilutions of the primary?

just out of curiosity, it has no bearing on your problem, why do you switch from milk to bsa and back to milk as your blocking agents? why not just stay with milk?