transfer of high molecular weight proteins - (Nov/14/2008 )

Hi
I am trying to probe for a high molecular weight complex but am not getting consistent detection. I use gradient gels and PVDF membrane for probing my 350kDa complex. Many times the background is immense though the bands are also present. Any suggestions please??

---

Make sure that it is running into gel and not stuck in the well.
-Gradient is good.
-Run for a longer time.
-Boil or not boil depending upon whatever works.

Make sure of complete transfer.
-PVDF is good.
-confirm by Staining the gel. PonceauS for membrane.
-Use thinner gel.
-Longer transfer time.
-May think of adding extra SDS to transfer buffer.
-Increased Methanol in transfer buffer may also help.

If transfer is complete, the background problem is a as good as any westerns.
-Refer to discussions on how to reduce background.

---

Blot cool (4 degrees C) and long (overnight -24 hours). It is best if you try several different times to see what works best for you.

---

Hi,

I am working with a high molecular weight protein (MW 512kDa). For protein detection, I tried 5% SDS PAGE gel but the protein got stuck at the interface between the stacking and the running gel. I am using 1% SDS (TGS based) for running gel and 1% SDS and 20% methanol in the transfer buffer. If any one has experience working with such a high molecular weight proteins, please give me suggestions.

---

Seems kind of obvious but you don't have to use a stacker. They aren't absolutely necessary, especially for high MW proteins, though they do make small proteins run more evenly. I would also have said that 5% was a bit high for a 500 kDa protein, you may need to switch to some other gel system.

I would also lower the methanol concentration in your transfer, it reduces the mobility during transfer.

---

reduce the sds in your transfer buffer. more than 0.05% will interfere with binding.

the 20% methanol will help strip sds from the protein to aid binding, you can leave it alone if you want (you can extend transfer time, if necessary).

we used to run a 3.5-5% gradient gel for high mw proteins (with a 8M-0 urea gradient, to sharpen the bands).

---

Oh great, many huge protein fans here. I also need your help

I try to detect a 310 kDa protein. Up till now I used 6% resolving gel, 5% stacking gel and a Tris-glycine buffer system.
I stained the gel and it seems that there are proteins > 250 kDa on my gel (that's the largest protein of my marker). I didn't stain the PVDF membrane after transfer as I read this might interfere with detection afterwards (right? wrong?), but I will do it next time. I had problems with blocking and I got a very nice secondary antibody binding up to 250 kDa, but not beyond. So I think my main problem is the transfer.

You mentioned not using stacking gels - does this also apply if I can't use gradient gels? Would you suggest 5% or 6% gels? Or even less?

For the transfer I used a semi-dry blotting unit, but as I read that this might be suboptimal, I ordered a tank blot unit. Do you have any suggestions for a good transfer buffer? Is SDS necessary in the transfer buffer? Mine has just methanol, but no SDS.

And one question for blocking: We usually use 5% BSA in TBS-T. In the data sheet of my primary antibody they suggest 5% milk powder in TBS-T. How do I sterilze this solution? I was told not to autoclave it, but filtration was...a bit....ineffective


Thanks a lot
Vista

---

if you start with 5-6% in separating gel you do not need a stacking gel; if used it should be lower than <5%;

you need a reference protein >/= 310 kDa;

use 0.037-0.05%SDS

why using sterile milk solution? through it away after usage...

---

I'm still waiting for the new blotting unit, but then I will try a 6% gel and a buffer with SDS.

Reference protein....hm.... I just want to show size differences and the second form of the protein is in the range of my marker and I have a positive control (cell line).

I thought sterile milk solution might be better if there's anything inside the powder (fibres or whatever) that might interfere the blot. But you're right, I will just prepare it fresh, that's the easiest way.

Thanks!

---

you should still filter (doesn't have to be sterile) to remove particulates when you prepare solution from powder.

---