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Questions related from Robert Adamu Shey
Hello everyone, we are characterizing some proteins in the context of vaccine development and we are interested in determining there are any epitopes that may rather lead to immune evasion. Don't...
04 April 2019 3,023 3 View
I want to purify IgG4 antibodies from immunized rabbits. I have tried to look for protocols to no avail. Commercial kits on the other hand exist for humans and a few other animals but I have not...
04 April 2019 10,092 3 View
Hello everyone, so I expressed this protein and I am trying to further purify using SEC and still having the impurities in the fraction I expected to have the protein. How do I eliminate these. I...
04 April 2019 7,647 3 View
I am doing RNA, DNA and protein extraction from the same sample and my protocol recommends this water saturated phenol:chloroform mixture. I am not not very versed with how to go about with the...
04 April 2018 4,386 8 View
Hello everyone, I am trying to do some hands-on bioinformatics and will be grateful to get your orientation.
01 January 2018 1,418 5 View
Hello everyone, I did 3D structure prediction (using I-TASSER) for a protein and submitted the best model for structure refinement (with GalaxyRefine). Unfortunately, I don't have a very good...
12 December 2017 1,307 0 View
Hello everyone, I am about doing an antigen capture ELISA and a little confused about which buffer can be used in absorbing the capture antibody to the microplates. I am using Nunc MaxiSorb 96...
11 November 2017 2,928 3 View
Hello everyone, So I planned this ELISA to do, unfortunately I coated plates and for some reason I am not able to continue with the experiment as planned. So basically, instead of coating...
10 October 2017 9,317 4 View
Hello everyone, I wish to express a gene cloned in pGEX5x3 through auto-induction and compare with the normal IPTG induction. Any one has experience doing autoinduction with pGEX vectors?
09 September 2017 6,340 0 View
I am working with a pET30a+ construct and my protein is C-terminal His-tagged. My protein is forming inclusion bodies and I need to get a protocol for isolation of a pure protein from this. An...
08 August 2017 2,044 4 View
I wish to find out if all secreted proteins have a signal peptide. Also, if yes, are these signal peptides always cut?
08 August 2017 9,964 2 View
I have serum samples in 50% glycerol and I want to detect by Western blots the presence of specific antigens in these serum samples? However, diluting these samples 1:2 and running on a PAGE gels...
07 July 2017 4,078 3 View
I loaded a 200ng of protein/lane for detection by WB. Unfortunately, Ponsceau S staining didn't show the proteins. What's the sensitivity of Ponsceau S? Can I procced with Western blots!?
07 July 2017 3,664 8 View
Hello everyone, I want to do ELISA to detect IgE in serum against an antigen I an working with and some one indicated that abundant proteins like IgG and HSA could either block or lead to...
07 July 2017 7,766 0 View
Hello everyone, I am doing lots of ELISAs and I am using carbonate buffer at pH 9.6 for coating. However, I wanted to find out whether using 50mM and 100mM carbonate can cause a difference in the...
07 July 2017 5,649 5 View
I working with protein that forms dimers and tetramers in the non-reduced state. I have to use it for immunological assays and I wondering whether these oligomers will not affect the signal I...
07 July 2017 8,952 6 View
I want to draw a phylogenetics tree using a protein I am working with and its homologs using MEGA7. After the alignment using MUSCLE or CLUSTALW, there are so many options on the MEGA program for...
07 July 2017 5,468 5 View
I am working with a recombinant protein from a parasitic nematode we expressed in bacteria and we have raised antibodies (affinity purified) against this protein. However, we have tried doing...
06 June 2017 4,318 7 View
Hello everyone, I am working with a difficult-to-express proteins and have changed expression host and cloning strategies a number of times. Finally, I was able to get something with cloning in...
10 October 2016 4,881 2 View
I am purifying a protein from bacteria and don't seem to get anything from the Western blots using anti-6xHis. Unfortunately, even the positive control doesn't give a signal and I have used this...
09 September 2016 1,865 4 View
I am working with a GPCR and intend to mass express the N-terminal which is like 224aas for immunological assays. Initially we cloned it in pET15b and put a 6xHis tag at the C-terminal (27kDa) but...
09 September 2016 7,821 3 View
Hello everyone, I am working on expressing a his-tagged 27.7 kDa protein (with 250 amino acids and 7 Cys residues) in BL 21 (DE3) cells using the pET15b plasmid. Unfortunately, getting a good...
07 July 2016 4,784 4 View
I have a protein contamination my purification on Nickel columns and we need a really pure protein to immunise animals. How do, I proceed with this. Thanks
07 July 2016 6,415 10 View
Hello everyone, I am working with a GPCR which seems to be very problematic: close homologs in Uniprot are not anotated and with respect to class some servers predict it to belong to Class A,...
05 May 2016 8,391 4 View
Hello everyone, I need some help here. I am interested in detecting a biomarker in urine. However, I have never worked with urine and so I will need you advice on how to process the samples. I...
03 March 2016 9,754 1 View
Hello everyone, I am doing western blots to detect a particular protein in crude worm extracts of a tropical nematode using antibodies raised in rabbits against a peptide from this protein....
02 February 2016 7,238 8 View
I am working with protein whose molecular weight is 23.6kDa. Upon induction and separation on a 12% SDS PAGE gel, I however am having the band at about 20kDa. I am using the precision plus...
11 November 2015 3,430 5 View
I am working with a parasite who total protein contents I have to analyse both by PAGE and Western blot. Can anyone suggest a protocol for going about this. I will be grateful. Thanks
10 October 2015 3,624 2 View
Dear All,I seem to have lost the plasmid DNA of one of my clones for protein expression.However, I do have a glycerol stock of BL21(DE3) cells transformed with the clone. Would it possible to a...
08 August 2015 4,576 4 View
Hello everyone, I am doing a batch-purification of a His-tagged protein using the His Select Ni Affinity gel and seem to losing my protein during the washes. The protein is present in the...
08 August 2015 3,566 8 View
Hello everyone, I am working with a 25 kDa protein which is 6xHis tagged. I have the gene coding for the protein in pET15b and expression is being done in BL 21 DE3. I tried purification and had...
07 July 2015 5,978 6 View
I am working on expressing a protein that has a C-terminal His tag. I want to find out whether anti 6xHis antibody and anti-6x C-terminal His antibodies will give me different specificities.
07 July 2015 6,240 4 View
Hello everyone, I am working on a GPCR which we initially failed to express in HEK 293 cells. Following consultations with some colleagues we switched to CHO K1cells but we detected proteins of...
06 June 2015 1,322 6 View
I have a protein, I am expressing in CHO cells ans it has the 6 His tag. I did the western blots and there is so much unspecific binding. The primary antibodies are raised in mouse. Could this be...
05 May 2015 3,512 3 View
I'm dealing HEK 293T cells for protein expression. How is many generations the maximum for passaging? Many thanks.
05 May 2015 9,867 4 View
I am working on a GPCR which we have predicted bioinformatically to be expressed in the endoplasmic reticulum. We have transfected HEK 293 cells and we are harvesting cells (24 hours, 48 hours, 72...
03 March 2015 2,594 4 View
I have a GPCR that I am working on. I have used a number of databases to classify it by bioinformatics but the webservers seem to be "confused" about it's class. I have used GPCR-CA for example...
03 March 2015 9,019 3 View
I am working on GPCR that has been predicted by bioinformatic analyses to be of Class A. Using UniprotKb indicates that it an intimal thickness related protein. However, I really do not have much...
03 March 2015 7,054 3 View
I am working with a putative protein whose homologues in the databases are predicted to be intimal thickness related receptors. However, I do not know much about such and very few papers are...
02 February 2015 9,109 0 View
I have 3 synthetic peptides that I am using for ELISA: analysis indicate that all of the have good water solubility. They have net charges of -1, -3 and -1.9 at pH 7 with corresponding PIs of...
02 February 2015 633 4 View
I am doing an ELISA to help me determine the immunogenicity of some synthetic peptides I am working for diagnosis. I have two sets of sera from patients with different but related disease and the...
02 February 2015 4,875 8 View
I have a PCR product that is 700bp with Nco I and Eco R I sites the 5' and 3' ends respectively. I need to insert this into pET28a+ and pET32a+ and need to digest the PCR product. How do I...
10 October 2014 2,921 23 View
Hello everyone; I have a issue that is seeming to be very challenging: I have to cleave an insert from a recombinant plasmid into pQE60. I have done some analysis on Webcutter and discovered that...
08 August 2014 772 4 View
I have a protein characterising and it happens to be a GPCR from bioinformatic analyses of the Class A. I have the gene coding for it in pET 15b vector and have tried to express it by IPTG...
04 April 2014 6,784 3 View
We trying to clone and express a GPCR in BL21-DE3-pLysS- bacteria using a pET15b vector, but we have been unable to induce our protein with IPTG. Can anyone suggest a means of overcoming this problem?
03 March 2014 6,648 9 View
Plasmid stability
03 March 2014 2,043 0 View
I need to do a molecular and functional characterisation of a novel antigen, in silico. Like structure, function, antigenicity and others.
02 February 2014 8,024 3 View
I am working on amplifying and mass expressing a plasmid in bacteria using a pET 15b vector but I have not been able to achieve this. I have tried many options but the results are still negative....
02 February 2014 4,622 2 View
