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Questions related to Protein Purification Techniques
Hi all, My western blot profile always show 2 clear bands at 110kDa and 75 kDa, no matter which secondary antibody I am using, be it rabbit and mouse secondary antibody. This problem will only...
04 April 2016 3,118 4 View
plz suggest the protocol for protein extraction from Natural gums. thank you
04 April 2016 5,066 2 View
I have a protein cloned in either pGEX-6P1 or pQE30 vector. It is not known to me. I am trying to induce the vector in XL1blue cells. how would I be able to verify which vector is it without...
04 April 2016 1,069 4 View
I have a protein that has about 120 kDa, I wanted to produce recombinant protein. Firstly I transformed my plasmid into competent cells and tried to have induction for this protein by using...
04 April 2016 7,978 4 View
My student and I tray to obtains Fc and Fab fragments from whole human IgG subclass whit this experimental conditions: ratio papain / Ab (3% ) (w/w), reaction time (24h), final volume (150µL) and...
04 April 2016 4,367 5 View
Hi everyone, I have a question related to the dissolution of a lyophilized synthetic peptide. I have a protein with a positive net charge, 23kDa, with Cysteines which I have tried to dissolve in...
04 April 2016 9,989 4 View
I just did a total RNA extraction using TRIZOL and the 260/230 values of all samples is between 0.50 and 0.90! I also noticed that for some of them the pellet did not dissolve completely. Also, I...
04 April 2016 5,656 7 View
Dear fellow scientists, I stumbled upon literature stating that the commonly used phosphotyrosyl phosphatase inhibitor sodium orthovanadate is potently chelated by EDTA. This may be a problem when...
04 April 2016 1,239 4 View
I have small volumes of protein samples that were precipitated by ammonium sulfate then dialyzed. Now I want to concentrate the protein samples, the method I have found so far are centrifugal...
04 April 2016 3,293 2 View
given that the samples were received with a membranous residue which have then dissolved after adding a lysis buffer. Anyone have faced the same issue? Is a membranous residue an indication of a...
04 April 2016 6,220 1 View
There are several papers where people have used perchlorate/ACN combination in HPLC to resolve peptides. However, it is reported that perchlorate in presence of acetonitrile forms free radicals,...
04 April 2016 6,797 1 View
My protein of Interest is of 12kDa (expected size). If I run it on a gel I get it around 18kDa. But in a reduced condition, It appears at 15kDa. Can anyone help me understand why is this...
04 April 2016 4,872 3 View
Hi All, I have been getting an unusual distinct band(size ~3kb) hanging down at the end of my gel with my genomic DNA. I was curious to find out what that is. My initial suspect was that it is a...
03 March 2016 2,540 3 View
I am interested in performing a western blot on cells that I have grown in Matrigel. I have read different protocols on recovering the cells from the matrix, including use of ice-cold PBS, Corning...
03 March 2016 6,018 1 View
Hello all I am currently working on FOX protein transcription factors, mainly FOXC2 and FOXF2. I have been facing some inconsistencies with Western bolts so far, any recommendations for robust...
03 March 2016 1,813 1 View
We performed WB but we got only single band from on 110KDa instead of 45kDa, with no other non-specific bands? We are looking for Gi protein receptors. 1.Is anyone working on tissue got this...
03 March 2016 5,062 5 View
Number of islets/condition protocol of protein extraction from pancreatic islet
03 March 2016 445 2 View
We have isolated total protein from small intestine of mouse using traditional RIPA buffer with 5-10X protease inhibitors (Roche) as well as with 2X Halt (Pierce). The lysate is perfect in terms...
03 March 2016 4,618 9 View
Dear all, I want to precipitate protein from supernatant of bacteria in culture and my question is whenever i do ammonium sulfate fraction i get floating pellet but, not getting settled pellet....
03 March 2016 1,481 7 View
I have been taking blood samples from Wistar Rats and I had some problems with plasma collecting. After centrifugation, plasma still showing a pale red. I am using a decapitation prodecure in...
03 March 2016 9,279 3 View
Hi All, I am trying to use DLS to verify that I am successfully reducing disulfide bonds responsible for the accumulation of mAbs in solution, yielding single uncoupled antibodies. Has anybody...
03 March 2016 3,811 5 View
Dear colleagues, We've tried to perform Co-immunoprecipitation on human cell line samples. After the Co-IP, we ran gels, excise individual bands of interest for MALDI-TOF/TOF protein...
03 March 2016 272 5 View
I have been working with A549 & HEK-293T cells and trying to do protein localization studies. I have conjugated my protein with His-GFP and the control is only His-GFP. I do find GFP signal in...
03 March 2016 6,973 5 View
We currently use Whatman 800µl UNIFILTER GF/F 96-well plates for purifying DNA under the Qiagen DNeasy tissue extraction protocol. We have commonly observed holes forming in the filter membrane...
03 March 2016 6,726 1 View