3 replies to this topic

[jojoziggy]

I am trying to detect a protein using a custom (peptide) antibody.  The protein is quite small (15kD or so).  On previous attempts I've found no band of the correct (small) size, but several nonspecific much larger bands do light up if I develop long enough.

I have had some anecdotal advice from 1 person about running/transferring/hybridizing small proteins to improve my results, but I'd like to hear if there's consensus from this forum on what actually works best!  My initial runs were run with 15% Acryl-Bis gel, 0.45 PVDF, transferred at 0.4A for 2 hr in our standard PVDF transfer buffer.

What I've heard:
Running:
use a gradient gel
or, use a high percentage gel (15% acryl-bis)

Transfer:
Use smaller pore size e.g. ~0.2um (makes sense, so the protein doesn't just float through!)
Use buffer w/o SDS (?? where can I find some buffer recipes ??)
Transfer for a "shorter" time
Transfer at a "lower" voltage
Use a dry transfer instead of a wet transfer

(I wasn't able to get a clear answer about what "short" time and "low" voltage actually meant.)

I also have heard (anecdotally) that protein pI can affect whether my transfer will work with a given buffer but not how!  And this isn't even mentioned in a bunch of Western "beginner's guides" that I have read.  Do I have to make sure my protein's pI is more acidic than the pH of the buffer?  Or more basic?

I find Westerns frustrating because there doesn't seem to be anything about the protocols that's empirically determined.  All I hear is "well... try a lot of conditions and maybe one of them will work"!  I find this scientifically lazy and I want some answers darn it!    

[jojoziggy]

any ideas?

[mdfenko]

what is your standard pvdf transfer buffer?

are you running the protein on sds-page?

have you stained a strip from your gel or a parallel gel to ensure that your protein ran properly?

15kDa protein is small (for purposes of page and transfer). 0.2um pvdf is recommended.

transfer buffer may have a small amount of sds in it to facilitate transfer of large proteins. this is generally unnecessary for small proteins.

shorter time and lower voltage may be necessary because you don't want to push too hard or too long or you will push the protein through the membrane and into the filter paper backing (you can add another membrane to back up the first to confirm this). by using the smaller pore membrane you may be able to maintain the transfer conditions that you normally use.

for small proteins it doesn't really matter if you do a wet or semi-dry transfer. just follow the instructions that come with the apparatus (or access them from the manufacturer's website if they are not readily available).

we often use tris-tricine sds-page for smaller proteins. resolution is better in that range.

[mightymouse]

You've probably already fixed this.  But for everyone else's reference, I was having trouble transferring myc tagged calmodulin (a highly charged small protein) and indeed it can pass through the membrane.  I used 2mM CaCl2 in my PVDF transfer buffer and transferred for 30min-1hour and got great results.  I got my protocol from this paper:

Calcium ion improves electrophoretic transfer of calmodulin and other small proteins
Thomas A. McKeonCorresponding Author Contact Information and Marian L. Lyman
Analytical Biochemistry Volume 193, Issue 1, 15 February 1991, Pages 125-130