How to express a mammalian transmembrane protein on gram negative bacterial surface?

We are trying to express a recombinant protein that is made of two different subunits. We have a plasmid with the alpha subunit and another with the beta subunit, but they are derived from the same plasmid (pcDNA3.1). We want to co-transfect them both at the same time to the cells, but we don't know if using the same promoter sequence for each subunit can impact the expression. Is it better to clone one of the subunits in different empty backbone?

I have been facing the issue of False negative results in both Dengue IgG & IgM assays. I have tried various buffer compositions with commonly known blockers to resolve sample interference but unable to resolve the false negative issue.

Hello,

I am working on a recombinant protein named Brolucizumab. the protein is designed as a monomer with a molecular weight of 26kd. It has 4 cysteines that form 2 s-s bind. I have a problem with this protein when I solubilize and Refold it. I solubilized it with 8M urea, tris50mM, and EDTA 10mM. For reducing the disulfide bond fresh dtt 10mM was used. after the protein was solubilized the solution was transferred to the refolding buffer in a ratio of 1/10. refolding components are L-Arg, Sorbitol, Tris, EDTA, urea 2M, and Cysteine/Cystine. Everything is fine so far. The problem is that when I solubilize Brolucizumab, ،The proteins tend to form dimers and trimers. the problem was solved by adding ddt in solubilization but after refolding you can see dimers and trimers. I monitored all of the steps by sds-page in both forms with and without BME.

at this time I don't have any idea how to solve my problem. can anyone help me with this problem?

Thanks.

If a protein locates at the inner membrane of mitochondria, and then it will be cleaved and released to intermembrane space of mitochondria, what is the fate of that part of protein still locate at the inner membrane?

I am trying to isolate BAC plasmid of size 36kb. I tried with Qiagen Miniprep kit and Zymo BAC purification kit and ran on 0.5% gel. The bands can be seen on the gel but no bands were after I did restriction digestion with 4-cutter enzyme. I also tried ethanol precipitation but there is thick band of 250kb size of I don't know what. What might be going wrong?

I have an insert size of 900 bp, trying to clone it in pET28a vector. But after every the transformation when i am doing colony pcr and digestion check i can only get the fragment of 500 bp. Why is it so? The restriction sites are BamHI and SacI and the gene doesn't have internal sites of these, which i could know by sequencing. So why is it so. Do i need to subclone instead of directly trying to clone in pET28a plasmid?

Hello Scientist.

I am currently doing qPCR to detect apoptotic gene expression in cell lines. After incubation with treatment, do i need to collect both dead and live cells OR only live cells to proceed with RNA extraction & qPCR?

Thank you in advance.

I was working on a gene construct synthesized in pet29 vector as a clone. Primers were prepared and optimized with gene at Tm 58 degrees. Once primers were optimized, I carried out transformation in expression vector and checked colony PCR with the same set of primers. After some time, I needed to conduct PCR for the same gene again for TA cloning and repeated BL21 transformation but issues occured. My primers that were previously optimized didn't work on the same gene on the mentioned Tm. After numerous trouble-shootings, I decided to check either the problem has appeared in my gene construct or not. I checked my commercially synthesized cloned gene on agarose gel in the intact and digested form and there was no band of gene once visualized. Is there any chance that my clone is destroyed by nucleases? What can be the reason for such conditions? It will be a great help if you can guide me

Hi,

I am carrying out the MTT assay using BJ-5ta fibroblasts cell line to evaluate the cytotoxicity of the nanocomposite hydrogels. I want guidance about deciding the controls in the experiment.

blank: media+MTT solution (to cancel the background noise)

positive control: Cells+media+MTT solution

control hydrogel (containing no nanoparticles)

hydrogels (containing different concentrations of the nanoparticles)

negative control : here I am confused and need guidance, whether it should be DMSO+cells+MTT solution

I will be grateful for your help!