small proteins wont separate!!!!!! Helpp!!

[annie02]

Hi,
I have been having the same problem with my SDS-PAGE gels in which the small proteins would not separate.
After coomassie blue staining. You can see that the ladder only goes as small as 29kDa. There are three more ladder marks below that (22, 14, 10.5kDa) which just won’t separate even when I run till the bottom of the gel.

This problem suddenly appeared about 3 weeks ago after I made new running buffer and separating buffer, however I have remade them again and the problem still persists.

I ran the gel at 200v for the stacking, and 100v for the separating.

I have tried pre-running the gel at 150v for 20mins before I load the samples. This way, the ladders do separate past 22kDa, however, the stained protein bands are too blurry.

Details

Ladder: Tris-Glycine 15%

10X Running buffer (4L):
120.00 g of Tris
576.00g of glycine
40.0g of SDS
pH to 8.3 using HCl
(dilute it to 1X to use)

10% Acryl Separating Gel recipe (for 2 gels):

5.0ml 40% Acryl
5.0ml 4X Separating gel
10ml ddH2O
10ul TEMED
75ul 10% APS

Stacking Gel recipe (for 2 gels):

0.5ml 40% acryl
1.25ml 4x stacking buffer
3.22ml ddH2O
10ul TEMED
20ul 10% APS

4x separating buffer:
91.00g of Tris
2.00g of SDS
pH to 8.7 using HCl

4X stacking buffer:
30.25g of Tris
2.00g of SDS
pH to 6.8 with HCl

Please give me some suggestions to solve this problem!! I am desperate!!

[thejoint]

if you want better resolution of small protein you have to make a 12.5 o 15% ACRYLAMIDE GEL.....

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