5 replies to this topic

[ale83t]

Hi! I'm new to the forum and I've an huge question: I'm working with the tat protein of HIV-1 virus and I need to transfer it to a PVDF membrane so I can make a western blotting, but I have problems to get it transferred to the membrane (it never transfers!!) using typical parameters.
Is there anyone that work with tat (or not...all help is appreciated! ) and can help me?

Thanks for any reply!

[bob1]

What is your protocol?  If you can post it, then maybe we can help a little more specifically than just listing the usual transfer/western problems.

[ale83t]

Ok. First of all i must tell you that i use a semi-dry system.
I use two buffers:
Buffer A: Tris 12.5 mM, Glycine 86 mM, Methanol 10%, SDS 0.02%
Buffer B: Tris 20 mM, NaCl 0.5 M pH 8

After the SDS-Page, I put the gel and the membrane in the Buffer A for 10 minutes, and then i assembly the sandwich using also filter paper wetted with buffer A as sayed by thenmanufacturer of the semi-dry system.
Then, i begin the transfer using a Current of 100 mA for about 35-45 minutes.
After the transfer, i wash the membrane 2 or 3 times (3-4 minutes every wash step) in Buffer B and then block it with Buffer B + 20 mg/ml of BSA for 1 hour.
After the blocking, there are some steps of washing with buffer B and then i incubate the membrane with the primary antibody in buffer B for 1 hour, wash the membrane and then incubate with the secondary AB.

I think that is a transfer problem because after the transfer step i've coloured the gel with comassie and there was all the Tat, but only a few quantity of the MW markers was transferred.
I hope it will helps you.

[bsengez]

I'm also working with HIV proteins and I had the same problem twice. can you find a solution?

ale83t, on Sep 15 2009, 02:45 AM, said:

Ok. First of all i must tell you that i use a semi-dry system.
I use two buffers:
Buffer A: Tris 12.5 mM, Glycine 86 mM, Methanol 10%, SDS 0.02%
Buffer B: Tris 20 mM, NaCl 0.5 M pH 8

After the SDS-Page, I put the gel and the membrane in the Buffer A for 10 minutes, and then i assembly the sandwich using also filter paper wetted with buffer A as sayed by thenmanufacturer of the semi-dry system.
Then, i begin the transfer using a Current of 100 mA for about 35-45 minutes.
After the transfer, i wash the membrane 2 or 3 times (3-4 minutes every wash step) in Buffer B and then block it with Buffer B + 20 mg/ml of BSA for 1 hour.
After the blocking, there are some steps of washing with buffer B and then i incubate the membrane with the primary antibody in buffer B for 1 hour, wash the membrane and then incubate with the secondary AB.

I think that is a transfer problem because after the transfer step i've coloured the gel with comassie and there was all the Tat, but only a few quantity of the MW markers was transferred.
I hope it will helps you.

[almost a doctor]

Are you pre-wetting the membrane in 100% Methanol before soaking in buffer A?   You need to do this with PVDF. Soak in 100% methanol and you'll see it become almost translucent, and then soak in buffer A (or water) until it sinks.  

Not sure if that's your problem as you say your markers transfer, but if is a tricky transfection it might help anyway.  

Also, you may want to try discontinuous transfer buffer, have a look at this: http://www.bio-medic...SD-Cell-1181-1/

SDS and methanol kind of contradict each other, but they are both needed.

hope this helps, good luck.

[cupidstunt]

100mA is a bit high I think. I usually use 30-40mA. But since your gel still has the protein on it that maybe a lesser problem. Have you tried Ponceau staining your PVDF after transfer?