Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!

research_freak's Content

There have been 11 items by research_freak (Search limited from 23-February 19)


Sort by                Order  

#67310 Protein precipitates upon dialysis

Posted by research_freak on 19 April 2010 - 12:56 PM in Protein Expression and Purification

Also, just for fun, check the pI of your protein and make sure the pH of your buffers is right for the pI.
You could also try different pH in your search for optimum conditions.


Denny,

Thanks so much!! Actually, I'm trying to purify a novel protein that was discovered in our lab. Expasy has a tool that characterizes proteins for its stability based on its amino acid sequence. It characterizes our protein as being unstable. Also, the theoretical pI of our protein is reported to be 8.40. I've heard that there are different programs to calculate theoretical pIs of proteins. What are the other programs that could help in identifying the theoretical pI?



#66089 GST-tagged protein binds to column but is not eluted

Posted by research_freak on 09 April 2010 - 06:07 AM in Protein Expression and Purification

Hey Guys,

I noticed that most of my GST-tagged protein comes into the soluble fraction during large-scale expression. When I tried to purify it on glutathione beads, most of my protein remains bound to the beads but fails to elute. Is there a way I can elute all of my protein?

Any advice is greatly appreciated.



#64796 Protein precipitates upon dialysis

Posted by research_freak on 30 March 2010 - 09:41 AM in Protein Expression and Purification

Guys,

I'm trying to purify my GST-tagged protein. My protein is about 55 kD in size, combined with GST which is 26 kD results in 81kD of my GST-tagged protein. It forms inclusion bodies, so I tried to solubilize it in 8M Urea. Next , I tried to dialyze it in PBS, however, my protein precipitates.

There are only 5 cysteine residues and I doubt if there are disulfide bonds. Any idea, why the protein precipitates?



#62832 Cellular proteins in medium

Posted by research_freak on 16 March 2010 - 05:52 AM in Tissue and Cell Culture

Hey guys!

I work with fibroblasts. Last week I had treated hepatic stellate cells with some drugs. I also had a control that was not treated with any drug. I had to analyze the medium and lysates from these cells. I first washed cells with serum free medium, and then added 0.5ml serum free medium exactly to each well containing cells. I usually incubate for 3 hours, and then collect the medium to probe for proteins. After preparing the medium, I also prepared cell lysates.

Now I had to probe both the medium and lysates to determine collagen and fibronectin protein levels. Along with these proteins, I also probe the lysates for tubulin and alpha-smooth muscle actin. Now, both these proteins are exclusively cellular proteins. Accidently, I happened to probe the medium for these proteins and detected a strong signal for these proteins in western blot analysis. Even the control cells showed a strong signal.

Does anyone have any idea as to how tubulin and alpha-smooth muscle actin - both exclusively cytosolic proteins could have gotten into the medium? I did not detect any visible signs of cell lysis upon examination under the microscope. I did not find collagen and fibronectin in the medium.

I am assuming that if the serum free medium i used was not warm enough, it could have resulted in cell shock and caused cell lysis which could explain how these proteins could be detected in the medium. However, if that's the case, I wonder why I couldn't find collagen and fibronectin? Now, both of these are very large proteins and our drug is seen to specifically target these proteins.

I appreciate all your input as to how and why I find these exclusively cytosolic proteins - tubulin and smooth muscle actin in the medium? What could have gone wrong? Has anyone ever experienced something like this?

THANKS!!!



#32889 Simple colorimetric assay for ribonucleotide reductase

Posted by research_freak on 13 August 2009 - 08:21 AM in Biochemistry

Hey guys,

I wanted to know if there is any simple colorimetric assay for detection of ribonucleotide reductase activity. I found some papers where they have used radiolabelled dCDP as a substrate, but I wanted to know if there was an alternative to this method.

Thanks!



#23704 Agarose gel electrophoresis troubleshooting

Posted by research_freak on 07 May 2009 - 09:22 AM in Electrophoresis

Always make sure you use fresh buffer everytime you run a gel (or at least when you're running important samples).
If the buffer is reused often, it is not of much good.



#23460 Sources of Contamination in plasmid DNA Mini-Preps

Posted by research_freak on 05 May 2009 - 04:42 AM in Molecular Biology

I'm not sure either, but I think that ssDNA as well as fragmented DNA have different absorption characteristics...so I wonder if that could possibly mess with the readings at 0.D. 260 nm....



#23369 Sources of Contamination in plasmid DNA Mini-Preps

Posted by research_freak on 04 May 2009 - 05:42 AM in Molecular Biology

I usually get a concentration between 100-500 ng/ul.

I thought that the high DNA concentration made sense initially since I had a lot of cells. But apparently my PI. thinks otherwise.

Also, I think that since the DNA binding column has a certain binding capacity, I wonder if something went wrong at this step...



#23366 Sources of Contamination in plasmid DNA Mini-Preps

Posted by research_freak on 04 May 2009 - 04:59 AM in Molecular Biology

I think it was contamination from the DNA prep and not from other contaminating organisms.

I used GENCATCH Plasmid DNA miniprep Kit.



#23365 Sources of Contamination in plasmid DNA Mini-Preps

Posted by research_freak on 04 May 2009 - 04:56 AM in Molecular Biology

I thank all of you for replying to my post!

Well, I measured the concentration using a nanodrop at O.D. 260 nm. Although the concentration should be between 100-500 ng/ul, I had a concentration of about 4500ng/ul in one tube and 2500 ng/ul in another tube!

I should have run this on the gel, but I had to hand it over to my P.I. immediately. :)
He was really not happy about it. Although I tried to ask other senior grad students regarding what could have gone wrong, they too said that possibly DNA must have gotten degraded and that could be the possbile reason for such a high concentration.

But I'm still shocked!



#23216 Sources of Contamination in plasmid DNA Mini-Preps

Posted by research_freak on 01 May 2009 - 09:47 AM in Molecular Biology

Hi,

I am a new grad student. Although I have done DNA mini-preps for about 2 weeks now, I've never had problems until this morning.

I did an E.Coli transformation yesterday, incubated my plates at 37oC and found a good growth of colonies. I then scraped all the cells from the plates and performed a mini-prep using a plasmid DNA extraction kit. Since there were a lot of cells, the initial pellet was very thick. Also, when I added the lysis and neutralization buffer, there was a large amount of white precipitate.

The issue is that I got a ridiculously high concentration of DNA in both my samples, which I assume to be a contamination.

Although I worked like any other day, I wonder what are the possible sources of contamination? I took all the precautions one needs to take while performing mini-preps..

Can someone please help me? :huh:

Thanks




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.