|Register||Search||Today's Posts||Mark Forums Read|
|SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.|
| ||LinkBack||Thread Tools||Display Modes|
small proteins wont separate!!!!!! Helpp!!
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.
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
75ul 10% APS
Stacking Gel recipe (for 2 gels):
0.5ml 40% acryl
1.25ml 4x stacking buffer
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!!
Re: small proteins wont separate!!!!!! Helpp!!
if you want better resolution of small protein you have to make a 12.5 o 15% ACRYLAMIDE GEL.....
[Only registered users see links. ]
|helpp , proteins , sds page , separate , small , small protein , troubleshoot|
|Thread||Thread Starter||Forum||Replies||Last Post|
|Human Cytome Project - Update 24 Jan. 2005||Peter Van Osta||Cell Biology and Cell Culture||1||08-01-2010 02:18 PM|
|Human Cytome Project - an idea - Update 19 April 2005||Peter Van Osta||Cell Biology and Cell Culture||1||06-01-2009 02:17 PM|
|SDS-PAGE with small proteins||Zhonglin Chai||Protocols and Methods Forum||0||11-30-2006 09:47 PM|
|SDS-PAGE with small proteins||Jess||Protocols and Methods Forum||0||11-30-2006 02:13 AM|
|Human Cytome Project - Update 6 Jan. 2005||Peter Van Osta||Cell Biology and Cell Culture||0||01-06-2005 10:18 AM|