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Questions related to Protein Purification Techniques
I am using gel filtration chromatography to analyze Hemoglobin at varying pHs. The results of the GFC at a pH of 7.4 showed that hemoglobin was in its dimer form (I was expecting to see Tetramer)....
05 May 2016 2,546 6 View
I am trying to extract protein from formalin fixed and paffin-embedded tumor tissues. The objective is then to perform western blotting of an membrane protein. Do you have any special...
05 May 2016 5,414 3 View
Is there any ready and checked ELISA/EIA/RIA kits or maybe I should think about chromatography assay?
05 May 2016 9,246 2 View
I have to purify a protein with 6xHis tag, but at the time of dissociating the protein from the resin (Ni-NTA) all protein remains in the resin. A feature of my protein is that it is rich in...
05 May 2016 4,894 6 View
Hello, I am doing disulfide bond detection by LC/MS-MS. Currently I met a very serious problem that my target protein has around 30 cysteins which can easily induce disulfide bond scrambling under...
05 May 2016 4,948 5 View
For an upcoming experiment I would have two kinds of DNA fragments in a sample after a particular reaction step: First there would be fragments of a length of approx. 170 bps and then there would...
05 May 2016 4,352 5 View
i am doing western blot for my research study and i am getting the black spots on the membrane after developing which indicate the binding of first anti-body to skim milk. how can i eliminate this...
05 May 2016 6,797 9 View
I run gel electrophoresis to confirm no presence of DNase contamination however my samples are remaining at the start of the gel. positive controls move as expected but my negative controls and...
05 May 2016 6,381 9 View
Hi, I am doing a gel shift assay to confirm that my recombinant protein will bind to DNA in vitro. I am using my rBAF180 and miniprepped pET30 vectors. I have tried undigested and digested...
05 May 2016 4,179 5 View
Hej Everybody. I am looking for to get answer how many 2DE gels I have to run for a pilot study in clinical proteomics? Should I pooled the sample and run three 2DE-gels or just run 3 good...
05 May 2016 6,720 4 View
Hello, I'm struggling with the unequal beta-actin expression level on western blot. I did the BCA twice and got the similar result, I'm sure the loading is correct. The loading amount should be 25...
05 May 2016 6,039 7 View
Hey, guys. I have always performed RNA extraction and gel electrophoresis of these RNAs. This time I got a surprise. My samples showed a band that had never appeared between 28S and 18S. Does...
05 May 2016 311 8 View
The sensorgram produced from the immobilization showing very strange behaviour, after activation with EDC/NHS, and upon injection of hAChE, the response begins to be negative, and then very slowly...
05 May 2016 483 1 View
Hello everyone, I have one research problem. I have to separate p53 (MW 53 Kda ) from total protein extracted from cancer cells and I have to study it by LC-MS. I have separated the 53 Kda...
05 May 2016 3,386 7 View
I've done Western blot analysis of Notch 4 proteins obtained from normal and carcinoma cell lines. Transfer is complete onto NC membrane. I've prepared new antibodies with dilutions 1:750. I tried...
05 May 2016 6,267 4 View
I have been trying to clone a 6.2Kb gene in CMV vector, but i am stuck at the initial stage itself. for PCR i have used LA Taq DNA polymerase/ Accu Taq LA DNA polymerase which produce intense...
05 May 2016 4,679 2 View
Hi all, I want to obtain a protein of interest without the first methionine and I would like to know if there is a expression protocol to optimize the methionine aminopeptidase of E. Coli or it is...
11 April 2016 426 3 View
Changing the polarity of the protein environment causes shift in the wave length of maximum absorbance. ie. less polar solvent causes red shift. This is usually along with a shift in molar...
04 April 2016 5,981 7 View
The peptide I am measuring is 30 residues long, and analogues will be lipidated at several different locations. We are currently worried that lipidation (with palmitate) will affect aqueous...
04 April 2016 8,692 2 View
I have a clone of a protein in pET21a and have tried expressing it in BL21DE3 cells and recently BL21DE3 star .On both occasions I have found loss of expression. I see a protein band with freshly...
04 April 2016 5,454 7 View
While using the 384 well plate replicator, what is the means of sterilization (disinfection) between plates to pick new clones from one plate to another? Anyone with experience on large BAC...
04 April 2016 8,632 2 View
I found the suitable procedure for the extraction of cellular debris from the glutaraldehyde fixed tissue. But I have a problem in the tissue homogenisation procedure theHEPES-sucrose buffer...
04 April 2016 4,253 5 View
I am trying to purify neutrophils from human buffy coat. I used Lympholyte-poly (CL5071), but red blood cells did not enter into the gradient and did not separate from neutrophils and...
04 April 2016 3,972 5 View
I need a reference / article saying something about how long period of time you need in total to perform an LLE procedure from plasma. Of course, I know a lot about LLE itself, but I need...
04 April 2016 1,512 2 View