Another Problem with Western blot. - (Sep/28/2004 )

Hi All,
I am desperate for help.
During SDS page:
I keep getting smear of my sample during loading. I loaded little sample of my protein of interest ( ~80kd) approx. 1000pg. in 10% seperating gel. I seems to have trouble getting a clear band. Could adding too much sample buffer be effecting the migration? As I used sample buffer to dilute my sample. ( When i used water the sample couldnt seems to sink into the well.

During Western blot:
I didnt even get a band or background signal on the X ray film?

my protocol was as below:
1. After transfering the proteins from the gel to nitrocellulose membrane at 40V overnight.
2. Stain the membrane with ponceu S.
3. Wash 2x with dH20
4. Wash 2x with TBS buffer
5. block with 5% milk for 1 hour at RT
6. Wash 2x with TBS buffer
7. Wash 1x with TBS T buffer
8. Primary AB (1/2000) for 2 hours
9. Wash 2x with TBS buffer
10. Wash 1x with TBS T buffer
11. Secondary Ab(1/2000) for 1 hour
12. React with chemiluminenece for 5 min ( blot membrane dry on both side)
13. Expose with X -RAy film..

No exposure.


Hopeful for a response?

hi jefften!

your protocol looks fine to me, seems to be ok.
but have you checked your system?
What I mean is, do you get signals when doing other kinds of Western with other Ab, but the same dry milk and ECL solutions? Have you ever gotten signals with the specific Ab you use here? How does the ponceau stain look like? Are the films of a charge you or anyone else has ever used successfully? Where the developer/fixer solutions changed or renewed lately?

You should inculde a positve control with your western to answer some of the questions...

mike

Thanks Mike. For the Reply.

Hmm.. I am still trying to troubleshoot the problems. Oh I think i made a mistake during my initial wash.. I might have wash with Di=I water for too long.. And Opps... Think i washed with TBS-T instead for TBS. before blocking.. That could have removed most of my proteins... I suspect..

Other people in the lab is using the same systems and membrane.. Many could get signal that they want.. so I believe theres something wrong with my technique..

Oh I am using the control... and still no band.. thats what made me really sad.

Im having the same proble...its really frustrating. Try using the reagents of a person in the lab that is getting it to work. Next, check your lysis conditions. Are you using fresh lysates? Work with someone elses protein see if you get the same results as they did.