Search found 14 matches

by Papaver
Wed May 30, 2012 10:32 am
Forum: Molecular Biology
Topic: Does urea disturb SDS interraction for SDS PAGE?
Replies: 1
Views: 1333

I did not have problems with SDS-PAGE and 8 M Urea in my samples...give it a try.
by Papaver
Thu Nov 17, 2011 4:07 pm
Forum: Molecular Biology
Topic: BN-PAGE of purified membrane associated protein
Replies: 7
Views: 3635

The pI is around 5.2.

Tomorrow I will run several native gels with different samples of the purified protein. I also did runs with the same sample but different amounts loaded onto the gel but as expected that wasn't helpful. In general I load around 10 - 20 µg.
by Papaver
Thu Nov 17, 2011 7:40 am
Forum: Molecular Biology
Topic: BN-PAGE of purified membrane associated protein
Replies: 7
Views: 3635

I guess you are talking about the sample buffer? The first time I used buffer containing coomassie brilliant blue (CBB) G250. Later I read that it is not necessary for purified proteins. The CBB of the cathode buffer is supposed to be enough to charge the protein. And in fact I do not see any differ...
by Papaver
Wed Nov 16, 2011 8:00 pm
Forum: Molecular Biology
Topic: BN-PAGE of purified membrane associated protein
Replies: 7
Views: 3635

That's funny :)

But can I do anything to improve the quality of my samples. Someway I hope it's the protocol and not the sample because then I cannot change that much. I was thinking to change the buffer (currently Epps buffer) but then I'm afraid my protein is going to precipitate.
by Papaver
Wed Nov 16, 2011 12:28 pm
Forum: Molecular Biology
Topic: BN-PAGE of purified membrane associated protein
Replies: 7
Views: 3635

BN-PAGE of purified membrane associated protein

Hello everyone, Since native page never worked with my protein I started to run BN-gels as it is supposed to be a good tool for hydrophobic proteins. I'm using gradient gels (3-16 %) because the protein seems to form high aggregated complexes. There is still one problem. I got protein smears so that...
by Papaver
Thu Oct 27, 2011 12:03 pm
Forum: Molecular Biology
Topic: PCR Accuracy?
Replies: 6
Views: 3902

The total error rate of Taq polymerase has been variously reported between 1 x 10-4 to 2 x 10-5 errors per base pair. The smaller your product is the better. If you really want to be sure that your sequence is correct then take a polymerase with proofreading activity.
by Papaver
Thu Oct 27, 2011 9:59 am
Forum: Molecular Biology
Topic: Removing MBP tag
Replies: 12
Views: 13490

Re: Removing MBP tag

Maybe it would be easier to produce your protein with no tag and just purify through several chromatographic steps The untagged protein is mostly found in inclusion bodies so it would be difficult to purify it. Anyway it might be worth trying it. The cells that you are using to express the protein ...
by Papaver
Fri Oct 21, 2011 12:17 pm
Forum: Molecular Biology
Topic: Removing MBP tag
Replies: 12
Views: 13490

It is!
So far, Strep- and GST-tag don't produce enough solube protein. I'm now trying to improve this. Not tried but in mind is NusA.
by Papaver
Fri Oct 21, 2011 11:58 am
Forum: Molecular Biology
Topic: Removing MBP tag
Replies: 12
Views: 13490

And, I already know that my protein highly aggregates. DLS measurement showed huge but stable aggregates which surprised us. This would explain why it is so hard to cut it completely or why so difficult to perform gel filtration (always found in void volume) and run native gels.
by Papaver
Fri Oct 21, 2011 11:23 am
Forum: Molecular Biology
Topic: Removing MBP tag
Replies: 12
Views: 13490

Hi, I've already tried everything: urea, guanidium, sodium deoxycholate, triton, tween, even acetonitrile (to separate the proteins). I also tried high salt (up to 700 mM NaCl), low and high pH (4.5/9). I know that it's partially uncut because I got three bands after cutting: one of MBP-Protein, one...
by Papaver
Tue Oct 11, 2011 9:04 am
Forum: Microbiology
Topic: Microbiology Book
Replies: 3
Views: 2603

For bachelor it's not easy to find a helpful textbook. Often you find more and better information in internet.
by Papaver
Tue Aug 16, 2011 2:19 pm
Forum: Molecular Biology
Topic: Removing MBP tag
Replies: 12
Views: 13490

Thanks for your reply. If I do the digestion outside I cannot reload the fraction onto the column because of the maltose in the purified fractions. And I cannot perform the digestion with the crude extract because of the mass of other proteins. So for me it's the best way to make it onto the amylose...