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protein elution from PAA gels - (Apr/24/2006 )

Hi all,

Can someone plz give me some protocol for elution of proteins from polyacrylamide gels.
mine is a hydrophobic protein 15.3 kDa, i need better recover coz i have it in small amounts.

Thanks!

-cheeztoast-

Hi,

I'm not sure what you're wanting to do with your protein, but here are some strategies that I've used for elution. Neither of these will produce proteins that are suitable for enzyme assays, but if you're just looking to use these to raise antibodies or perform mass spec analysis, they're just fine.

One thing you might want to consider first is changing your detergent from SDS to lithium docecyl sulfate. LiDS doesn't co-precipiate with proteins in acetone like SDS will, so you can remove the detergent in one step. Another thing to try is using a zinc stain (again, repacing SDS w/ LiDS...don't use the commercial zinc stain kits, as they have SDS...you'll need to make your own...don't worry, it's really easy). Zn stains are preferrable in this case, as they don't crosslink your protein to the acrylamide matrix, and so your recovery is much better. Just be sure to chelate the Zn ions w/ EDTA before performing elution.

ok...so now for the protocol...

run you LiDS PAGE gel (use LiDS in your sample and running buffers too), and do the Zn stain. (note, if you know where your protein is in the gel and it's nearly pure, you might not need to stain...simply use a razor and cut the area of the gel w/ your protein out). once you've cut your gel, de-stain for about 10 min, and rinse w/ ddH20 (or something similar). from here, you chop your gel piece up w/ a razor, or (if you have a large enough piece of gel) freeze it in liquid nitrogen and grind it to a powder in a mortar and pestle (be careful of sample loss here if you try this). elute the proteins O/N at 4C on an end over shaker using 50mM Tris, 50mM NaCl, 0.1% LiDS (or more, considering you're dealing w/ a hydrophobic protein) at about 3-4X volume of your gel pieces.

the next day, centrifuge the gel pieces and take the spt away. if you have enough protein, concentrate it by precipitation in 4X vol acetone. if not, concentrate in a microcon or some other sort of centrifugal concentration device (at 4C).

next, run out an aliquot of this on a SDS-PAGE gel to make sure you've actually recovered your protein.

it helps to start out w/ a large amount of protein (a preparative gel is an easy way to go), but if that's not possible, just be careful to avoid losing sample.

there are kits out there for this, but i've found this protocol gets better recovery.


i hope this helps.

if you have more questions, email me at jonmike.reed@gmail.com


quote name='cheeztoast' date='Apr 24 2006, 06:01 AM' post='49043']
Hi all,

Can someone plz give me some protocol for elution of proteins from polyacrylamide gels.
mine is a hydrophobic protein 15.3 kDa, i need better recover coz i have it in small amounts.

Thanks!
[/quote]

-johanski-

Actually i have an unidentified high mol wt impurity in my purified prep for the protein. if i can get it out i can run an IEF gel or an hplc or something to know what form of my protein it is. so yr protocl shud work fine.

Thanks much!
Abt LiDS.. i dont think i have it in the lab.. shal hunt for it tho. if not i can get it, but is thr a quicker option without it? getting it wud take time.

and if u can.. plz let me the procedures for Zn staining (the one that u follow) and what kind of EDTA chelation is need before elution.
Thanks again:)

[quote name='johanski' date='Apr 24 2006, 07:30 PM' post='49064'] Hi,

I'm not sure what you're wanting to do with your protein, but here are some strategies that I've used for elution. Neither of these will produce proteins that are suitable for enzyme assays, but if you're just looking to use these to raise antibodies or perform mass spec analysis, they're just fine.

One thing you might want to consider first is changing your detergent from SDS to lithium docecyl sulfate. LiDS doesn't co-precipiate with proteins in acetone like SDS will, so you can remove the detergent in one step. Another thing to try is using a zinc stain (again, repacing SDS w/ LiDS...don't use the commercial zinc stain kits, as they have SDS...you'll need to make your own...don't worry, it's really easy). Zn stains are preferrable in this case, as they don't crosslink your protein to the acrylamide matrix, and so your recovery is much better. Just be sure to chelate the Zn ions w/ EDTA before performing elution.

ok...so now for the protocol...

run you LiDS PAGE gel (use LiDS in your sample and running buffers too), and do the Zn stain. (note, if you know where your protein is in the gel and it's nearly pure, you might not need to stain...simply use a razor and cut the area of the gel w/ your protein out). once you've cut your gel, de-stain for about 10 min, and rinse w/ ddH20 (or something similar). from here, you chop your gel piece up w/ a razor, or (if you have a large enough piece of gel) freeze it in liquid nitrogen and grind it to a powder in a mortar and pestle (be careful of sample loss here if you try this). elute the proteins O/N at 4C on an end over shaker using 50mM Tris, 50mM NaCl, 0.1% LiDS (or more, considering you're dealing w/ a hydrophobic protein) at about 3-4X volume of your gel pieces.

the next day, centrifuge the gel pieces and take the spt away. if you have enough protein, concentrate it by precipitation in 4X vol acetone. if not, concentrate in a microcon or some other sort of centrifugal concentration device (at 4C).

next, run out an aliquot of this on a SDS-PAGE gel to make sure you've actually recovered your protein.

it helps to start out w/ a large amount of protein (a preparative gel is an easy way to go), but if that's not possible, just be careful to avoid losing sample.

there are kits out there for this, but i've found this protocol gets better recovery.


i hope this helps.

if you have more questions, email me at jonmike.reed@gmail.com


quote name='cheeztoast' date='Apr 24 2006, 06:01 AM' post='49043']
Hi all,

Can someone plz give me some protocol for elution of proteins from polyacrylamide gels.
mine is a hydrophobic protein 15.3 kDa, i need better recover coz i have it in small amounts.

Thanks!
[/quote]
[/quote]

-cheeztoast-