western blot looks strange - western blot looks very weird (Mar/28/2006 )

hello everyone

i made a few western blots and some of them look really weird and i dont know why. You can have a look at them at: fotos.web.de/jakob.schumacher/dr. I have very similiar results nearly every time, so it must be a systematic error.

blocking: 5% BSA 1h at room temperature
1 antibody: llama (tagged with myc)
2 antibody: Mouse anti Myc 1:1000 or 1:2000
3 antibody: Goat anti Mouse HRP 1:15000 (with 0,05% tween)
washing: 1% BSA
and i use the multiprotean scan from biorad

after the reblot of the third antibody (stripped with Sodium Acide for 30min.) it looks better but still not ok



can anybody help me?

thanks in advance jakob

Well, i can't login to the website to take a look on your blot

do you have any detergent in the wash buffer? might be a problem......try tween20

HI,
it is difficult to understnad wat is happening with your western blot with out looking at pictures, we cant login to ur site.
y dont u attach ur pics directly?

Hi

I don't think that this is a problem but I am thinking why do you use 3 antibodies?
You can use an Mouse anti-Myc-conjugated to HRP!

Paste the picture of your blot if you want our advice.

I looked at your pics... and for me looks more like a trap assay .
Same question, why 2 antibodies, what are you trying to detect and what its molecular weight?
For how long do you wash after each antibody incubation? seems that the HRP conjugated antibody binds very unspecifically, specially in those 2 black lanes.
Maybe you have to block again after putting second antibody if you dont have a secondary labeled with HRP.