|
11/28/2008 10:32 AM
|
|
11/11/2008 02:47 PM
|
|
11/11/2008 09:17 AM
|
|
9/30/2008 06:52 AM
|
|
9/29/2008 06:38 AM
|
|
9/20/2008 01:00 PM
|
|
9/4/2008 09:25 AM
|
|
8/27/2008 11:22 AM
|
|
8/9/2008 10:37 AM
|
|
8/6/2008 09:43 AM
|
|
7/21/2008 06:12 AM
|
|
7/21/2008 06:12 AM
|
|
7/21/2008 06:55 AM
|
|
7/21/2008 05:59 AM
|
|
7/8/2008 07:31 PM
|
|
7/2/2008 12:25 AM
|
|
5/9/2008 11:42 AM
|
|
4/26/2008 11:54 AM
|
|
1/28/2008 09:49 PM
|
|
1/19/2008 02:27 PM
|
|
12/20/2007 06:14 PM
|
|
11/16/2007 08:00 PM
|
|
11/5/2007 03:00 PM
|
|
10/25/2007 04:09 PM
|
|
10/25/2007 03:27 PM
|
|
10/10/2007 10:43 AM
|
|
10/9/2007 09:34 AM
|
|
9/27/2007 03:31 PM
|
|
9/17/2007 04:30 AM
|
|
9/13/2007 09:26 PM
|
|
no protein after dialysis [View Printable]
|
linamira
Group: Member
Posts: 4
Joined: Sep 16, 2008
|
DEAR SCIENTISTS,
I have expressed a rrcombinant protein in E.coli (with a high yield 25mg/ml cell culture)) and purified it using Ni chromatography and now, I am trying to concentrate my protein against PEG-8000 , when water get out of the dialysis bag , i washed the bag with 500 ul PBS and I musured directly the protein concentration using the bradford assay.the assay revealed that my protein concentration is undetectable (0mg/ml). any suggestins please!? |
.........................
|
| Posted Sep 16, 2008, 20:55 PM |
Last edited Sep 16, 2008, 20:57 PM by linamira |
|
|
Carson O Genic
Group: Member
Posts: 152
Joined: Jun 22, 2005
|
What size is the protein and what size is the membrane? |
.........................
|
| Posted Sep 17, 2008, 11:04 AM |
|
|
|
R Bishop
Group: Admin
Posts: 321
Joined: Jan 17, 2006
|
Good start Carson o Genic.
How do you know the concentration of your protein was 25mg/ml. That is incredibly high?
Further what type of dialysis tubing did you use? Brand? clips? etc.
Rb |
.........................
"To those who would tear the world down: We will defeat you. To those who seek peace and security: We support you. And to all those who have wondered if America's beacon still burns as bright: Tonight, we proved once more that the true strength of our nation comes not from the might of our arms or the scale of our wealth, but from the enduring power of our ideals: democracy, liberty, opportunity and unyielding hope."
-- Barack Obama, Nov. 4, 2008
|
| Posted Sep 18, 2008, 1:32 AM |
Last edited Sep 18, 2008, 1:34 AM by R Bishop |
|
|
linamira
Group: Member
Posts: 4
Joined: Sep 16, 2008
|
HI,
I am using a dialysis membran with 12000-14000 d cut-off , my protein is about 25 KD .The protein concentration before purification is very high but it decreses after purification and disapesrs after dialysis. I don t know where is the problem? |
.........................
|
| Posted Sep 18, 2008, 10:07 AM |
|
|
|
R Bishop
Group: Admin
Posts: 321
Joined: Jan 17, 2006
|
Hmm
several possibilitites.
1. your protein "disappears" so either its not their in the first place (check it by gel after purification, not Bradford).
2. you have some faulty dialysis tubing and its going through
Are you using clips and tying the ends of your tubes? or is it a product like the slide-a-lyzer from Pierce?
Here's a simple test. Find some Blue Dextran (> 100kD) put that in the dialysis tubing, dialyze it. if it comes through buy some new tubing.
3. it is sticking to the walls of the tubing - lack of detergent after dialysis may cause this.
Im betting on number 2.
Let us know after you check these items.
Good Luck
Rb
|
.........................
"To those who would tear the world down: We will defeat you. To those who seek peace and security: We support you. And to all those who have wondered if America's beacon still burns as bright: Tonight, we proved once more that the true strength of our nation comes not from the might of our arms or the scale of our wealth, but from the enduring power of our ideals: democracy, liberty, opportunity and unyielding hope."
-- Barack Obama, Nov. 4, 2008
|
| Posted Sep 18, 2008, 10:58 AM |
|
|
|
|
top of page
|
  
|
Forum Jump
|
|
Refer a Friend
Link To Us
Bookmark Us