Way too many bands on western blot

[es0teric]

Hi all,

I have recently been doing western blots on yeast whole lysates, looking for my proteins of interest using a commericially available monoclonal primary antibody (ascites). My blots are lighting up beautifully with low background signal BUT there are way too many bands (I mean like WAY too many, it looks more like an SDS page gel than a western blot! With discrete bands to boot.)

My primary antibodies are from mouse and I have tried using GE healthsciences' sheep anti-mouse-HRP antibodies as secondary.

I have done some troubleshooting and found that the secondary antibodies SEEM to be unspecific as the blots now light up in the same fashion without primary antibodies (negative control was primary antibodies only, didn't light up). I have tried to fix this by using a different secondary antibody from goat this time and the effect is still the same, many discrete bands lighting up (a control was used here - secondary goat antibodies only).

What is the chances of 2 different secondary antibodies binding unspecifically??

primary antibody conc. 1:4000
secondary antibody conc 1:5000

EXASPERATED. PLEASE HELP!

[missoliverita]

I have the same problem, please let me know if you figure this out, my film also looked like an SDS gel. I used the protocol given to me by another PhD, she said to use a 1:2000 dilution.

Im going to try washing longer first, and also I dilute 1:2500 hihihi