Search found 14 matches

by dhkwak
Fri Jan 07, 2011 2:22 am
Forum: Molecular Biology
Topic: Electrophoresis of ionic complexes
Replies: 6
Views: 5018

Re: Electrophoresis of ionic complexes

Thanks, canalon. That sounds a very good idea. Actually, only one of our polymers consists of a protonated amine group, while the others are quarternized (i.e., RN(CH3)+). However, it might be a good idea to try. As you may have guessed, this siRNA-polymer complex is for physiological use, so I'm no...
by dhkwak
Wed Jan 05, 2011 5:55 pm
Forum: Molecular Biology
Topic: Electrophoresis of ionic complexes
Replies: 6
Views: 5018

Re: Electrophoresis of ionic complexes

Thanks, JackBean. Typically, native electrophoresis is done at 100V or higher (buffer is virtually the same, but there are no reducing agents or detergents). Just in case anyone is wondering, the cationic and anionic moiety is protonated amine (polymer) and phosphate (siRNA), respectively. I can't i...
by dhkwak
Tue Jan 04, 2011 9:42 pm
Forum: Molecular Biology
Topic: Electrophoresis of ionic complexes
Replies: 6
Views: 5018

Electrophoresis of ionic complexes

I've been performing agarose gels to determine the complexation between two ionic species: one is a polymer approximately 10nm in diameter; the other is 19bp siRNA. In brief, I've been running my gels at 100V, and I do not want to see these two species separate; instead, I want to see how they compl...
by dhkwak
Tue Apr 20, 2010 4:56 pm
Forum: Molecular Biology
Topic: Issues with Bradford Assay
Replies: 3
Views: 3839

Hey guys, I had run some small experiments to see what I was doing wrong and though I am still in the process of more definitively figuring out what had happened, here are my thoughts: The CBB-G250 I had used is in the green form. CBB-G250 can take on three forms: acidic (red-brown), neutral (green)...
by dhkwak
Mon Apr 19, 2010 7:56 pm
Forum: Molecular Biology
Topic: Issues with Bradford Assay
Replies: 3
Views: 3839

Hey JackBean, Thanks for the reply. I was too quick to ask about orthophosphoric acid vs. other forms of phosphoric acid, as they are essentially the same. I'm sorry about that. I don't suspect very much precipitation and I did not see signs of such. I accidentally wrote 100-200 ug per mL, when it i...
by dhkwak
Mon Apr 19, 2010 7:05 pm
Forum: Molecular Biology
Topic: Issues with Bradford Assay
Replies: 3
Views: 3839

Issues with Bradford Assay

Hey all, I have had some troubles with the Bradford method and wanted to know if anyone who has sufficient experience with this procedure could provide some insight. First, I had produced my own Coomassie dye (using CBB G250) solution and had noticed that in solution (before being bound to protein),...
by dhkwak
Sun Mar 14, 2010 9:06 am
Forum: Molecular Biology
Topic: Precipitation of BSA
Replies: 1
Views: 3129

Precipitation of BSA

Hey guys, I have precipitated BSA using by: (i) adding cold, dry acetone in 5 or more quantities per protein solution, (ii) incubating at -20 Celsius for at least an hour, and (iii) centrifuging at 13,000 g for 15 min and removing the supernatant. I wanted to know if anyone has had issues with using...
by dhkwak
Thu Mar 11, 2010 8:44 am
Forum: Molecular Biology
Topic: When is sterilization necessary?
Replies: 1
Views: 1124

When is sterilization necessary?

Hey all, I was wondering when is sterilization necessary? I am working on a project that is analyzing the binding of a protein to a polymeric surface and was informed that it could simply be done in the chemical hood or the bench and not the biological hood. I wanted to know if it is commonly a prob...
by dhkwak
Mon Mar 08, 2010 5:03 pm
Forum: Molecular Biology
Topic: Running SDS PAGE for the first time!
Replies: 9
Views: 7517

I am wondering if there is a rule of thumb for the amount of SDS to use per protein. I have often read that a 4:1 (SDS:protein) ratio is sufficient but am unsure if this refers to concentration, weight, or otherwise.

Any help would be great, thanks!
by dhkwak
Tue Feb 23, 2010 8:24 am
Forum: Molecular Biology
Topic: Running SDS PAGE for the first time!
Replies: 9
Views: 7517

Hey guys, I've run several gels by now and am starting to feel more and more confident with my technique. However, I am still having difficulty effectively separating proteins of fairly similar MW. In short, I have a protein (BSA) and that same protein cross-linked with a polymer (about 3kDa). The M...
by dhkwak
Tue Feb 16, 2010 5:43 pm
Forum: Molecular Biology
Topic: Running SDS gels
Replies: 2
Views: 1300

I think it's fair that you would like them to use TEM in the hood. I wouldn't imagine they would be doing that intentionally. I don't know exactly what your relationship with your colleagues is like, but maybe you can quietly take one of them aside and explain your condition and politely ask them to...
by dhkwak
Wed Feb 03, 2010 4:23 pm
Forum: Molecular Biology
Topic: Running SDS PAGE for the first time!
Replies: 9
Views: 7517

Hey Guys, I really appreciate the help. I'm debating as to whether load approximately 50ng or 50ug. I am thinking about measuring concentration of my protein after running it on the gel, however I need to be sure that my protein separates cleanly (although I highly suspect it will not). My analyte i...