Phosphorylation of kinase domain in bacterial expression system?

Hello. I want to crystallize kinase domain of a protein in active form. The active form of the domain has specifically phosphorylated tyrosine residues. However, I am not sure if I can do so in a...

01 February 2020 7,443 5 View

Double band after protein purification?

I have purified a protein, however I am getting double band on SDS-PAGE. Even after FPLC the double band doesn't resolve and become more prominent. I have checked through Mass spec that both the...

06 July 2019 6,634 10 View

Tool to understand the interactome of a kinase domain of a protein?

Is their any online bioinformatic tool available through which I can understand or know the interacting partners of a kinase domain of the protein I am studying? I wish to study interactome of...

06 July 2019 5,996 6 View

Double bands in SDS-PAGE gel?

I am purifying a protein expressed in pLysS cells. The construct is in pET28 vector and having His tag with TEV cleavage site . Now when I ran the SDS-PAGE gels after TEV cleavage and...

03 April 2019 7,380 16 View

No cleavage site in C terminal having His tag?

In my construct I have two His tags, one at N-term and another at C-term. There is a TEV site between the N-term His tag and my gene but no cleavage site for the C-term. So, do I need to add...

03 April 2019 2,231 8 View

Transformation in Rosetta2?

I am not getting colonies when I had transformed my construct in rosetta 2 cells whereas transformation worked in Bl21-pLyse cells. I had used the same heat shock protocol for both. The one change...

01 February 2019 2,053 11 View

Protocol for lysate preparation from mouse tissues for western blotting ?

01 February 2017 5,221 6 View

Marker for determining genetic relatedness and distance?

I want to study genetic relatedness among fresh water fishes, so which molecular marker would be best for this study?

07 August 2016 6,154 2 View

Control for oral cancer?

06 July 2016 1,252 4 View

Assays or protocol for TORC signalling?

Does any one have any protocol or assays by which I could study signalling profile of tor gene during various stress conditions in yeast?

06 July 2016 9,871 0 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

I want to performe MALDI-TOF MS for bacterial identification, I would appreciate it if anyone can tell me the matrix solution preparation protocol ?

based on what I've studied on multiple papers, for peptide and protein identification Alpha-cyano-4-hydroxycinnamic acid (CHCA) is an appropriate matrix to use, I also know that the CHCA powder...

26 February 2021 6,799 3 View

Difficulty in cloning of 1kb gene into vector?

I am facing difficulties in cloning a 1kb gene into a vector (pJIT163). I have my gene on interest (GOI) in pUC57 and want to clone in pJIT163 using SalI and BamHI restriction sites. I am getting...

25 February 2021 3,221 7 View

Universal bacterial primers targeting a mRNA?

Hi, I'm looking for universal primers targeting a bacterial mRNA to quatify bacterial load by qPCR in a complexe mixture (eukaryote and prokaryote RNA). Ribosomal RNA being depleted, I absolutly...

22 February 2021 9,969 3 View

Covid 19 Pandemic ' impact on digital transformation?

Covid 19 Pandemic and digital transformation

22 February 2021 3,351 5 View

Does anyone have any ideas why drug-resistant malaria is increasing in Southeast Asia even when those drugs are no longer used?

When dealing with bacterial toxins, there are three varieties of microbes that develop in your petri dish: toxic, resistant, and susceptible. Initially, there are only toxic and vulnerable...

21 February 2021 6,084 5 View

I can detect my protein but the encoding DNA sequence has stop codons within. Should I ignore the stop codons?

I constructed a synthetic DNA library (scFv, VH-VL orientation) with a 3' reporter and 8x histag. I cloned and expressed this gene following which I performed an ELISA. The ELISA results suggested...

19 February 2021 7,006 5 View