Protein ladder - (Sep/18/2008 )

Hi,
I'm currently trying to optimise western blots but the protein I am trying to identify is quite large-about 300kda.
I'm looking for a prestained protein marker to run alongside my samples on SDS-PAGE. I've been trying to look for a suitable ladder but I'm getting a bit confused. Does the type of ladder I buy depend on the type of gel I use (I use a Tris-HCl gel)?
Can anyone recommend any suitable high-weight protein markers?
Thanks:)

You can use every denatured protein marker suitable for SDS-PAGE. Here the reference proteins in the marker are denatured and reduced like your proteins of interest. These are the most common markers sold. It is harder to find markers for native PAGE. But I think it will be a bit more difficult to get a marker which comprises your high mw.

Thanks for your reply
So does this mean that the likes of this ladder would be suitable?
http://tools.invitrogen.com/content/sfs/ma...stained_man.pdf

Even though it just says "Suitable for NuPAGE® Novex®, Tris-Glycine, and Tricine Gels" (while my gel is Tris-HCL pH 8.8)?
Sorry this is probably a really stupid question, but I'm just really confused about the whole thing

Anyone have any idea at all?

Pre-stained standards are good for assessing transfer efficiency during westerns, but are not so good for estimating the size of your protein. This is because of the dye molecules attached to the proteins -- the dye molecules are pH dependent, and thus the standards will migrate at different apparent molecular weights under different conditions. See the tables for each standard listed here.

There is also a selection guide for Invitrogen standards here.

A Tris-HCL gel is just the original Laemmli gel, around since the 1970's. It usually has a stacking gel of pH 6.8 and a separating gel of pH 8.8. This gel uses chloride as the mobile anion; other gel chemistries use other anions.

There's no reason why any ladder would not work with this gel system. However, as I said, the migration of the pre-stained standards is sensitive to pH, so you'd need to calibrate your pre-stained standard with a non-stained standard to get the apparent molecular weights as they behave in your system. This only comes into play if you're using the standards to find the molecular weight of your protein. The ladder will be separated fine, and it will transfer fine -- so if all you're using it for is to tell when your gel has run far enough and whether transfer has occurred sufficiently, any pre-stained ladder will be fine. You might get some different colors due to variation in pH, but other than that, you're ok.

as homebrew said, the tris-hcl gel referred to in your product pamphlet is a denaturing (sds) gel.

if you want to run a native gel then you can find non-denatured standards at ge healthcare:

elecrophoresis standards

note, the lmw standards are non-denatured (even though the product name says "sds-lmw", it contains neither sds nor reducing agent - you have to add it yourself if you use them with sds-page).

the high mw standard (native) is what you are probably looking for.