Transfer of large proteins (>500 kD) to nitrocellulose. How?

[Anton Tutter]

Gunther Rezniczek wrote:
> 
> Dear collegues:
> 
> For some time now I have - unsuccessfully - tried to
> transfer large proteins (MW range 500-700 kDa) from SDS-PAGE
> gels (4.5%) onto nitrocellulose membranes.
> I have tried semi-dry and wet blotting procedures with
> varying buffer and current conditions.
> 
> Has anybody succeed in transfer of such large proteins?
> Any ideas, suggestions or complete protocols?
> 


Gunther,

I routinely transfer proteins to nitrocellulose.  I have had no problem 
transferring large MW proteins (>300kD) successfully when I use the 
following conditions:

4.5 - 5% gel
50mA current per mini-gel for transfer
1 hr transfer, re-wet w/ transfer buffer
1 hr transfer, re-wet w/ transfer buffer
1 hr final transfer

total = 3hr transfer

Keep in mind that some transfer buffer recipes call for SDS.  Sometimes, 
depending on the protein, this may or may not aid in the transfer.

Here's the recipe I use:

    2.93g Glycine (Free Acid, not HCl)
				5.81g Tris-Base
				2.55ml 10% SDS
				200ml MeOH

				q.s. to 1L with H2O


Good luck, and let me know if it helps!!!

Anton Tutter
Salk Institute
La Jolla, CA