I am relatively new to western botting. When I first learned this procedure I used a 7.5% NEXT gel buffer blend, without the addition of a stacking gel. This is my first time running a SDS-PAGE with a stacking gel.
I ran a SDS-PAGE today with the gels I casted myself. My protein of interest is Spinophilin, which is expressed at ~120kDa. I used a 4% stacking gel and a 7.5% resolving gel. I ran the gel at a constant 175V, however after 4 hours, majority of my prestained protein marker (Novex Sharp Pre-stained Protein Standard LC5800) was still within the stacking gel.
However, my protein samples seemed to be appropriately migrating through the stacking and resolving gel. I noticed there was about 2cm of space between the bottom of the well and the top of the resolving gel, instead of 1cm, which is the recommended.
Will this extra cm cause my protein marker to become delayed in entering the resolving gel?
The recipes for my gels are listed below:
7.5% Resolving Gel - 10ml
- 4.85mL ddH2O
- 2.5mL Tris-HCl (1.5M) pH 8.8
- 2.5mL Acrylamide
- 100ul 10% SDS
- 50ul 10% APS
- 15ul TEMED
4% Stacking gel - 10ml
- 3.35mL ddH2O
- 2.5mL Tris-HCl (0.5M) pH 6.8
- 4mL Acrlyamide
- 100ul 10% SDS
- 50ul 10% APS
- 15ul TEMED
I checked the pH of both Tris-HCl and they are correct.
Any help or suggestions is greatly appreciated!
It looks like something is wrong with your recipes:
Your Resolving Gel and your Stacking Gel solutions have the same volume - 10 ml.
In your Resolving Gel (7.5%) you used 2.5 ml of Acrylamide (I guess that was a mixture of Acrylamide and Bis-Acrylamide), but for your Stacking Gel (4%) you used 4 ml Acrylamide. The Resolving Gel should always have the higher concentration of Acrylamide than the Stacking Gel. Try to use the concentrations of Acrylamide vice a versa (2.5 ml for Stacking Gel and 4 ml for Resolving gel) and change the water volume added and everything should be fine.
I would also recommend to check the recipe for your SDS-Loading Buffer (especially pH) that you used to dissolve your samples. The fact that the commercial protein standards migrated different way than your samples might indicate that something was wrong with your SDS-Sample Buffer.
The distance between the bottom of the well and the top of the resolving gel is not that crucial. I would not worry about that.
I agree with Victor, your recipe suggests you have reversed the resolving and stacking gels.
Also, in future, pictures of the final gel are very useful in diagnosing problems.
Use the attached chart/table for preparing gel, sample buffer and tank buffer, hope you get good gel. All the best
Dear, Certainly some thing is wrong, prepare fresh stocks, you may see alternates and tested recipes at the following link chapters 8-11:
Book Labtop for Microbiology Laboratory
Check out this working recipies for resolving and stacking gel
4 mL ddH2O
2.5 mL Tris-HCl (1.5M) pH 8.8
3.3mL Acrylamide
100ul 10% SDS
10ul 10% APS
4 ul TEMED
5% Stacking gel - 3 ml (is enough)
- 2.1 mL ddH2O
- 3.8 mL Tris-HCl (1M) pH 6.8
- 5 mL Acrlyamide
- 30 ul 10% SDS
- 30 ul 10% APS
- 3 ul TEMED
Hope this will help.
I agree with all those reply and there is a mistake in gel preparation but you say that your protein is separated but your presained marker is still in stacking gel . I think some time this might happen if air bubbles formed within the well where you loaded marker and that causes hindrance in protein mobility.
I agreed with @Rakesh Kumar. It do happens if there is bubbles in the well
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