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I am a cell and molecular biologist interested in deciphering the function of proteins that have an impact for human health but have not been yet fully characterized. I have participated in studies across a variety of topics (the malaria parasite (Plasmodium spp.), the ubiquitin system and the regulation of insulin secretion) and have therefore experience with different cellular models as well as useful techniques for the study of protein function.
Short‐chain 3‐hydroxyacyl‐CoA dehydrogenase (SCHAD), encoded by the HADH gene, is a ubiquitously expressed mitochondrial enzyme involved in fatty acid oxidation. This protein also plays a role in insulin secretion as recessive HADH mutations cause congenital hyperinsulinism of infancy (CHI) via loss of an inhibitory interaction with glutamate dehyd...
The proteasome is the major non-lysosomal proteolytic machine in cells that, through degradation of ubiquitylated substrates, regulates virtually all cellular functions. Numerous accessory proteins influence the activity of the proteasome by recruiting or deubiquitylating proteasomal substrates, or by maintaining the integrity of the complex. Here...
A proper cellular adaptation to low oxygen levels is essential for processes such as development, growth, metabolism, and angiogenesis. The response to decrease in oxygen supply, referred to as hypoxia, is also involved in numerous human diseases including cancer, inflammatory conditions, and vascular disease. The hypoxia-inducible factor 1-α (HIF-...
I will do an enzymatic assay using a-ketoglutarate as substrate and I am wondering about the proper way to prepare and store this compound. As long as I could find out, it would be OK to dissolve it in water and store it in aliquots at -20*C. Is that correct? What about the stability? for how long is should be good to use?
I am trying to produce my protein of interest fused to an N-terminal GST tag in BL-21 e.coli cells. In general, I can produce the protein but most of it remains in the pellet after cell lysis. I have tried plenty of conditions to improve the solubility but I have not succeeded. I only get small improvements and most of the protein remains in the pellet.
The conditions I have tried are the following:
* Different temperatures: 28'C / 15'C
* Additives: EtOH / Glycerol / Triton
* Different IPTG concentrations: 0,05mM - 1 mM
* Different times of induction: 0,5 - 16 hours
* Different lysis buffers: pH 5 - 9 / NaCl 50mM - 2 mM
* Different lysis methods: Lysis buffer for bacteria / Sonication / Sonication + Lysozyme / French press
Do you think there is something else worth trying?. According to ExPASy predictions my protein (including the GST tag) should be very soluble.
Hi! I am measuring the activity of my enzyme of interest through the decrease on the absorbance at 340 nm (to detect the disappearance of NADH). I am measuring every minute for 8 minutes, and I am using an amount of protein that produce a nearly straight line when plotted Time vs. Absorbance. I am confused regarding the way in which I should report my data, and what kind of calculations I can do and information I can get, according to the source of my enzyme (The main objective of my study is to compare the activity of different mutants):
* Total lysate from transfected cells, that over-express my enzyme of interest.
* Cell free transcription/translation system (TNT).
* Purified recombinant protein expressed in E. Coli.
My guess is that I can calculate different things when, for example I use the recombinant vs. the cell lysate. I also guess that these different systems to test the activity can be complementary to each other.
Besides getting answers to this question, I would be happy if you could recommend some literature that could help me to clarify and deepen my knowledge on these issues.
Hi! I am performing and enzymatic assay, where Acetoacetyl-CoA is the substrate. I have gotten the Acetoacetyl-CoA powder that SIGMA sells (5 mg). I think it would be easier to handle if I just make a solution with all the powder, and store it in aliquotes for later use. However, I am not sure which solvent should I use to dissolve Acetoacetyl CoA. The buffer for my enzymatic assay is a phosphate buffer pH 7. SIGMA only says that this compound is soluble on water, but I have read that the Acetoacetyl-CoA solution should be in a pH between 4 and 6 to be stable for several months. Any suggestions? I need to use a solvent that will not interfere with my enzymatic assay. Thanks! :)
I want to use it as a co-factor of an enzymatic reaction, to evaluate the enzymatic performance of the enzyme and the kinetics of the reaction. I have read that it can be very tricky to work with NADH as it is hygroscopic, can oxidize and can form inhibiting compounds when dissolved and stored. Is there perhaps any NADH that is sold in solution?.
I am trying to express my protein of interest in a TNT cell free expression system (Promega, L1170). My protein is tagged With V5 and His tags. The problem is when I run the samples in a western blot, though I can see a band of the proper molecular weight (about 37 kDa), I see a smear under it. I attach a picture so you can see what I mean. In the lines 2 and 3 I am using the anti HA ab, and in 4 and 5 the anti His. (The first one is a marker and in the last two I am using an antibody against the protein that does not seem to recognize my tagged protein very well).
I followed the instructions of the manufacturer. I also tried adding protease inhibitors, but I get the same results.
Anybody has any ideas?