In what circumstances we need to add antibiotics (eg. ampicillin) into LB broth and agar?

I'm working with an integral membrane protein that we want to express and purify for structure/function studies. The full-length gene is cloned into a pET vector with a C-terminal His6 tag. We get fantastic expression in E. coli C43(DE3), but when blotting lysates and crude membrane fractions, there is only weak blotting at the top of the gel/membrane.

When membranes are re-solubilized in detergent and assessed by SDS-PAGE, there is a large and prominent band just smaller than the expected size of the recombinant that does not bind a Ni column. Several prominent, high MW bands are present in crude membranes and to a lesser extent in detergent-reconstituted membranes. Digest and LC/MS of the soluble species confirms that it's the recombinant protein, but C-terminally truncated so lacking the tag. The truncated region contains an important active site motif, so the truncated form is also inactive in assays when purified.

I have a few ideas to obtain the full-length form (cell-free translation, denaturing purification) but was curious if anyone had experienced something similar and had used an approach to prevent truncation in a similar case. We can map the likely site of proteolysis from the LC/MS data, but not sure what to do with that information.

During writing a review, usually published articles are collected from the popular data source like PubMed, google scholar, Scopus etc.

My questions are

1. how we can confirm that all the articles that are published in a certain period (e.g.,2000 to 2020) are collected and considered in the sorting process(excluding and including criteria)?

2. When the articles are not in open access, then how can we minimize the challenges to understand the data for the metanalysis?

Hey everyone,

I’m relatively new to the field of mass spectrometry and proteomics and need some advice. I’m trying to run a recombinant protein on our mass spec. It should be a really straightforward protocol: aliquot out the protein and resuspend in buffer, reduce, alkylate, digest in solution, desalt, dry, resuspend in running solution, and run on the mass spec. But somehow, in the one step that I could possibly have loss - the desalting step - I get around 80% loss. The kit I use is made for the amount of protein I aliquot (20-50ug) so I have no idea why I’m losing so much.

If you guys have any advice for cleaning up recombinant protein digest specifically, I’d be very grateful. Thank you!

Hello!

I am performing a research on potato protoplast extraction and regeneration and am wondering if someone can help me getting the composition of the medium V-KM (Binding and Nehls 1977) used in protoplast culture. This medium is cited by different authors but I can't find its composition.

I think it is especially for moulds of the Aspergillus and Penicillium genera. I don't rmeember the reason! Do someone know? Thank you

#fungi #agar #inoculation #moulds #aspergillus #penicillium

Hi everyone. I would like to grow Fusarium oxysporum f.sp cubense TR4 on PDA media. I planned to add antibiotics to prevent bacterial contamination. So far as I searched, most people recommended streptomycin and chloramphenicol, which are not in my hand at the moment.

May I know if ampicillin or kanamycin can also be used for this purpose? If they do, what would be the appropriate working concentration?

Thank you!

I want to express a recombinant protein, but I am unable to obtain the mRNA sequence encoding this protein. Therefore, I intend to use a sequence available in a database. Thus, I would like to request the synthesis of a vector containing the gene sequence, but I am unsure whether to ask for the cloning of the entire mRNA sequence (1534 bp) or just the coding sequence (1220 bp). The CDS is directly embedded within the mRNA sequence, but there are nucleotides before and after the CDS.

Dear colleagues

Please suggest a comprehensive and complete protocol for the production and purification of bacterial recombinant protein in E.Coli BL21 strain.

Hi,

does anyone know of a database or method for predicting the expression of a recombinant protein in E. Coli which strain of E. coli is better to have the highest soluble expression of the protein predicted by the database, and which back-bone vector would be best for expression?

Thank you in advance for your help.