Western blotting problem - Protein and proteomic (Feb/26/2006 )
I have followed the western blot method based on some manual. I used PVDF membrane and ponseau S staining for 5 minutes, the blocking agent is 2.5% skim milk powder in TBST for 1 h. Then primary antibody Lyn is 1:500 concentration for overnight at 4 C without shaking. Then I used 1:5000 secondary antibory for 1 h, ECL A and B reagent 500 ml each and it was incubated for 2 minutes. Finally the X-ray film was exposed 10 minutes. I could not get any band. Please let me know, may i should alter some blotting step. please suggest me
regards
anbusamy
OK it's a naive question, but do you have a positive control, like recombinant protein. What's the concentration you used?
Thank you so much for your reply. I have used 20 ug protein. May i use the bitrocellulose membrane instead of PVDF membrane. I have also loaded positive control. If you have further idea, please let me know. Thanks in advance
Anbusamy
20 ug of protein ! (the protein recognized by the antigen?) It's quite a lot.
with WB you should detect 20 ng.
Usually I block in TBS + 3% non fat dry milk, at 4°C overnight, but 1 hour room temperature is enough. sometime milk is too strong, and I replace it by BSA.
Try to block in TBS + 3% BSA, either over-night, in the cold room (not necessary to shake) or 1h at RT, under gentle shaking.
If I were you I would incubate the antibody in TBS (without tween or triton : which one do you use?), plus 3% BSA, or even less : 0.3%. one hour, RT, with gentle agitation
Wash in TBST twice and once in TBS
Incubate the second antibody in TBS plus 3 % non fat dry milk (with ECL you should block with something strong like milk).
wash 3 times in TBST and twice in TBS
keep PVDF. with PVDF you get a stronger signal, but you might get more background compared to nitrocellulose
good luck
a little question, how have you tested your ab? With Dot blot? If your +control is negativ, its possible that your ab are too strong diluted?! 1:5000 is a lot!
It depends which antibodies you are using, but I would say that 1:5000 doesn't seem so much diluted...
I used HRP-anti-rabbit from Amersham diluted 1:30 000 (provider says 1:5000) and anti-mouse 1:20 000.
Now I'm using from santa cruz, diluted 1:10 000.
works fine.
but it's a good remark.
did you test your antibodies?
you could do dot blot just to check
Are you sure your blotting has been successful? You can use a reversible total protein stain from Pierce to check that you have protein (and equal loading) on your membrane. You can then remove the stain without affecting the Ab incubation steps.
Also, are you diluting your chemoluminescence reagents? Some protocols tell you to dilute 20-fold, etc but I used to just add 1 mL of each, mix them together and throw the whole two mL on to my blot.
Is your primary Ab at a high enough concentration. I've needed as much as 1:50 before for certain proteins. Finally, you haven't told us where your two aBs come from. Check you're not reacting an anti-mouse IgG with a rabbit polyclonal or something equally incompatible.