Problem choosing the right kind of Precast gels - (Mar/08/2006 )

Hi!

I have a problem deciding which type of precast gel provided by Bio rad would be suitable for my experiments.

The standard protocol I follow works fine. Just thought of saving time by using pre-cast gels.

The following are the ingredients I use for the standard protocol. Could someone advice me which is the type of pre cast gel I should opt for. I require a 7.5% gel.





Resolving Gel (7,5%)
3x 5x
H2O 9ml 15ml
Acrylamid(29:1)4,5ml 7,5ml
Temed 45ul 75ul
APS (10%) 67,5ul 112,5ul
(always fresh)
4x Lowerbuffer 4,5ml 7,5ml

Stacking Gel
1x 2x 3x 4x 5x
H2O 3,7ml 7,4ml 11,1ml 14,8ml 18,5ml
Acralamid (29:1) 0,8ml 1,6ml 2,4ml 3,2ml 4ml
TEMED 15ul 30ul 45ul 60ul 75ul
APS (10%) 20ul 40ul 60ul 80ul 100ul
(always fresh)
4x Upper Buffer 1,5ml 3ml 4,5ml 6ml 7,5ml





4x Lower Buffer

1,5M Tris-adjust HCl pH 8,8
1% SDS



4x Upper Buffer

500mM Tris-adust with HCl pH 6,8
0,4% SDS




TBST 10x

100mM Tris pH 8,0
9% NaCl
0,05% Tween 20 ‡ add just before use

(2L 200ml 1M Tris (pH 8.0)+180g NaCl)



Transfer Buffer

2L
36mM Glycine 5,8g
48mM Tris base 11,63g
0,037% SDS 7,4ml (10%SDS)
20%Methanol 400ml

The main things to think about when choosing a pre-cast gel is how well that gel will separate your proteins and standard. If you look at the Invotrogen catalogue they have a fantastic image which gives you an idea of how proteins are size separated by the different gel systems they offer. Some are better for small Mw proteins others for high Mw proteins. Some are good if you want to blot two proteins of quite different sizes on the same membrane.

In addition, the buffer system the gel is based on also affects its shelf life. Some gels only last for a month some will last for a year. Depends how quickly you'll get through them.#

As an example, I ran westerns on P-glycoprotein (172 kDa) using 7% Tris-Acetate pre-cast gels. This gave excellent resolution and separation of my markers as well.