5 replies to this topic

[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 and poteins only runs till ~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 run 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!!

[bob1]

Use a higher percentage gel - 15% should work.  10% will work for proteins in the range of 25-70 kDa, whereas 15 will work for 10-40 kDa.

[annie02]

bob1, on 24 May 2011 - 06:30 PM, said:

Use a higher percentage gel - 15% should work.  10% will work for proteins in the range of 25-70 kDa, whereas 15 will work for 10-40 kDa.

Thanks for the quick reply!

I have tried using a 12% gel, it did separate the proteins smaller than 29kDa, however, the smaller protein bands were blurry., and not as intense as the larger protein's bands. This way I cant compare their concentrations.

I have been using 10% gels for months and it would separate till 22kDa just fine, but now it wont. It must be something I am doing or using differently, but I just cant think of what. Is there other factors that could cause it to not separate?

[casandra]

annie02, on 25 May 2011 - 08:15 AM, said:

bob1, on 24 May 2011 - 06:30 PM, said:

Use a higher percentage gel - 15% should work.  10% will work for proteins in the range of 25-70 kDa, whereas 15 will work for 10-40 kDa.

Thanks for the quick reply!

I have tried using a 12% gel, it did separate the proteins smaller than 29kDa, however, the smaller protein bands were blurry., and not as intense as the larger protein's bands. This way I cant compare their concentrations.

I have been using 10% gels for months and it would separate till 22kDa just fine, but now it wont. It must be something I am doing or using differently, but I just cant think of what. Is there other factors that could cause it to not separate?

hi annie....so you don't really have a specific target protein/s but are looking to compare concentrations of several proteins in your sample from the high MW to the lowest? Coz you probably know that normally, the percentage of gels you'd use depends on the size of proteins you're looking for...i.e. the smaller proteins would have better resolution using a higher percentage of gels....

Edited by casandra, 25 May 2011 - 08:56 AM.

[bob1]

Westerns for small proteins are tricky as the proteins diffuse really fast - using a higher percent gel will slow this a bit.  Between finishing the run and setting up the transfer your gels should not be sitting around for more than a couple of minutes - 10 at most.

If you need to separate a wide range with good resolution, try gradient gels.

[mdfenko]

do not adjust the pH of the running buffer.