Western blot running and loading samples - (Jun/15/2010 )

Hi everyone!. This is my first post.

Well, i'm using Western blot and after using Semi-dry transfer I usually dye my nitrocellulose with Ponceau. Every Western I do, I have the same problem: protein below 30 Kda do not appear or are smeared
Please someone help me!

Ps: another question. Durind the loading of samples I have to diluite my protein in Laemmli. I usually do 1:1 diluition. Is it correct?

Thanks.

-GFAP-

GFAP on Jun 15 2010, 06:50 AM said:

What percentage is your gel? Did you tried to use a more higher-percentage gel (>12%?). Maybe even a Tris-Tricin buffer system?
How you dilute your sample in Laemmli-buffer depends on the concentration of the same. Is it 2x or 4x?

-snoopyx-

I have tried 15% and 18% but it's the same. My laemmli is 2X.

Thanks

-GFAP-

GFAP on Jun 17 2010, 08:13 AM said:

I have tried 15% and 18% but it's the same. My laemmli is 2X.

Thanks

Hi GFAP, welcome to the forum, I have a few questions for you:
Have you stained your gels before transfer to check your separation? How long do you electrophorese for? What voltage? How long do you transfer for? What voltage?

I think you should check your gels to confirm whether your problem is with the transfer or with the electrophoresis. If you see a smear you might be having problems with the separation. If you dont see bands at all, you might be transferring for too long so that the smaller MW proteins have gone past the membrane. You could put 2 membranes to check for this.

That's all I can think of right now as you've not given us much detail. Uh, also, what buffers are you using for EP and transfer?

Oh, if your buffer is 2x, a 1:1 dilution is good.

-almost a doctor-

Thank u almost a doctor for your reply!
Well, I'm sure that my problem is the separation. ..usually my electrophoresis is 200V for 50 minutes.No i'don't check the gel. How can I do it? I check nitrocellulose after the transfer with ponceau and I see smearing in the end. High molecular proteins run very well. After 30 kDa I see my proteins smearing.
My semi-dry transfer is 15V 25 minutes.
-My buffer 1X for ep is:

trizma 3.03g
glycine 14.4 g
sds10% 1g
H20 (volume 1 L)

-transfer 1X is:
trizma 3,03g
glycine 14,4g
methanol 200ml
h20 (1 L)

Thank you

Hi GFAP, welcome to the forum, I have a few questions for you:
Have you stained your gels before transfer to check your separation? How long do you electrophorese for? What voltage? How long do you transfer for? What voltage?

I think you should check your gels to confirm whether your problem is with the transfer or with the electrophoresis. If you see a smear you might be having problems with the separation. If you dont see bands at all, you might be transferring for too long so that the smaller MW proteins have gone past the membrane. You could put 2 membranes to check for this.

That's all I can think of right now as you've not given us much detail. Uh, also, what buffers are you using for EP and transfer?

Oh, if your buffer is 2x, a 1:1 dilution is good.

200V for 50 minutes!!! tat is too high!!!
u surely must be running the lower molecular weight proteins out of the gel!!!

does the corresponding mol weight in the marker lane stay inside the gel??!!

to check your gel, run a parallel gel (if your samples are not too precious, although you waste them if the gel doesn't run correctly) and stain it with coomassie blue or silver.

rather than run a 15 0r 18% laemmli gel you may want to consider a 10 or 12 or 15% tris-tricine gel (as pointed out in a previous post). it works especially well with low molecular weight proteins and peptides.