Google Sign in options
Remember me
This is not recommended for shared computers

Sign in anonymously
Don't add me to the active users list

Privacy Policy
Sign In
Create Account

Javascript Disabled Detected

You currently have javascript disabled. Several functions may not work. Please re-enable javascript to access full functionality.

Wickerl3732

Member Since 05 Aug 2011
Offline Last Active Feb 13 2013 04:05 AM

Find content

Community Stats

Group Active Members
Active Posts 10
Profile Views 269
Member Title member
Age 36 years old
Birthday November 16, 1976
Gender
Male
Location
BIDMC-Harvard Medical School
Hobbies
vascular biology

About me

My research interests
Interferon
vascular biology

Contact Information

0 Neutral

Friends

Wickerl3732 hasn't added any friends yet.

Topics I've Started

restricted PCR plasmid runs slower

12 February 2013 - 05:59 PM

Hi everyone.

I am preparing a PCR product insertion into my vector. It is 1080 bp product, and I am restricting at the 3' with HindIII to cut off 76 bp and on the 5' end I introduced a EcoRI restriction site with my forward primers, allowing me to cut off 11 bp.
But here is my problem: after restriction and analysis on an agarose gel I had the opposite which I would have expected. The 1080 bp PCR product runs at the right size, but after restriction with HindIII the band appears to be a little bit higher and after double digest with EcoRI, this band was even higher.
Do you guys have any reasonable explanation for this. I was guessing that the restriction enzyme might stick to the DNA, or that a conformational change may cause the higher band (?).
Paralleling the restriction of the PCR product I digested the plasmid with the very same restriction enzymes, and this digest was perfectly fine.

too much DNA on agarose gel

30 January 2013 - 07:04 PM

Hi to everyone.

for purification of my PCR product I was loading a too high DNA concentration on my preparative gel, and, as one would expect, it ran way too fast and the band appeared very low. However, when my student was asking me why DNA runs too fast if you load too much, I had no reasonable explanation.

Can anyone help me out here?

co-IP after ubiquitin cleavage

14 September 2012 - 08:35 AM

Hi,
I am persuing a finding, that protein A interacts with protein B and that this interaction is either dependent of k48 ubiquitination of on of these proteins or dependent on unlinked ubiquitin chains. To verify my assumption, I wanted to digest the cell lysate with isopeptidase T to cleave the ubiquitin chains, and I expect that my two protein of interest would not co-immunoprecipitate anymore.
However, activation of isopeptitase requires 10mM DTT, and the enzymatic reaction should be performed with at least 1 mM DTT. I guess that this concentration of reducing agent will disrupt my protein-protein interaction anyway.

Does anyone have any suggestions how I could analyze the dependence of two proteins on ubiquitination? I would highly appreciate your help!