Ankita Dutta

International AIDS Vaccine Initiative | IAVI · Department of Public Health

In hippocampal neurons if synaptic vesicle fusion is impaired why does mini IPSC amplitude remain same as control but the evoked IPSC is decreased? The miniIPSC frequency is decreased relatively to the control.

My protein shows intrinsic tryptophan flourescence. I used this property to study the lifetime properties. In the same context i performed acrylamide and KI based quenching methods to study accessibility of the probe in presence and absence of an inhibitor. In the protein without inhibitor I see gradual quenching of fluorescence as expected however, on addition of the inhibitor I see an erratic pattern. There is no consistent decrease in fluorescence intensity. I changed the quencher from acrylamide to potassium iodide (sodium thiosulphate) but it has not helped. I have to calculate the kq value. Please give suggestions to solve the issue. * protein samples were filtered after addition of inhibitor to remove any aggregates if formed.

Insert size: 1Kb

Vector : 4.5 Kb

Restriction enzyme sites : NdeI XhoI

Backbone was dephosphorylated with rSAP for 1 hr 37C heat inactivated later,

PCR product was gel eluted after digestion from pGEMT clone which had positive fall out.

260/230 ratio 2-2.2 ( good)

Ligation conditions: 16C overnight and 25C 8hrs, 1:3, 1:5 ratio Vector: Insert.

Transformation in DH5alpha ultracompetent cells efficiency 10^8

Colony PCR was done showed amplification- directly from plate, colonies were picked grown overnight- pellet was taken lysed by heating and then PCR was done to confirm (to prevent false positive results from ligated dna contamination)... either case the PCR results were positive, Non Template control doesnt show any amplification (no conatmination in the buffers or reagents)

Yet after 3hrs of double digestion the fall out is not seen at all on the gel. Changed the enzyme to confirm if the enzyme had an issue ...got similar results.

What could possibly go wrong... Please suggest. Incase any further details are required I would provide them. Thanks 

I want to try Molecular dynamics simulation of my protein with manganese(II) and Cobalt(II) ions. Can anyone tell me if the coordinates for the metal ions available for carrying out the same?

I have been trying to crystallize a protein that is functional with both Cobalt(II) and manganese (II) ions.The holoprotein has to be prepared by heat activation. For the same  protein the conditions with manganese (II) were known. I tried to use the reported conditions for manganese protein that is PEG3350(25%), 100mM Bis tris ph 5.5, 1mM MnCl2 and 15mM Guanidium HCl. Another way was to make the holoprotein and then use the same conditions to setup the drop. In either case the nucleation sets in 24 hrs and I get small needle like or plate shaped crystals at 16C. These further do not grow at all. I have tried variations of the PEG types and its concentrations, also variation in the buffer pH. The protein concentration is approx 6 mg/ml. Also, I had tried the Morpheus screen in which ethylene glycol condition gave some crystals which also did not grow in size. Hampton screen has so far not yielded any positive results. Another problem I am facing is that whenener I try to set up screens with Cobalt. The protein always precipitates whether I add CoCl2 in drop or make the holoprotein and then set up the drop. Can anyone please give me any suggestions on how to overcome any of the above two issues- firstly the further growth of the crystals with manganese and second the prevent the precipitation of protein with Cobalt. 

I am trying to express and purify a helicobacter protein. I had tried cloning and expression with CT his tag(pET21c+) but the protein was in hard inclusion bodies. So I changed my tag to GST (pGEX). Expression is fine but mostly protein is in the pellet. Can changing the bacterial growth medium from 2X YT to TB help? is there any other way to increase protein solubility and expression. I am using the Rosetta plysS strain for expression as it showed better expression than bl21plysS. 

I have a his tag protein in inclusion bodies that i purify in denatured state by affinity chromatography. I have standardized that 8M urea with or without DTT is giving me maximum solubilization of protein from inclusion bodies i n mini scale. But when I try the purification in large scale keeping ratio of Inclusion bodies and solubilization buffer same as in mini scale also keeping every other parameter same I am losing maximum protein in the inclusion body pellet. Also I have tried the 7M urea, 2M THIOUREA, 2% CHAPS, 5mM DTT combination. It is comparable to 8M urea in mini scale. I increased the volume of solubilization buffer as well but rather it lead to further loss of protein in washing steps. Should I change the denaturant to Guanidium hydrochloride?