Shilpa Sarkar

14 Questions 10 Answers 0 Followers

Questions related from Shilpa Sarkar

Can anyone suggest a protocol to express and purify G alpha from E.coli cells?

I have expressed a parasite G alpha protein seperately with a GST tag and his tag in E.coli BL21 cells and looking for purifying the active form. Some people have used AlCl3 and NaF in the...

25 December 2021 5,069 5 View

Can anyone suggest a protocol to purify active form of G alpha protein from bacteria?

I am heterologously expressing a G alpha protein along with GST tag in BL21. Can any one suggest a protocol to purify the active form of the protein which could be used for further experiments?

14 August 2021 9,744 1 View

How to validate a protein structure?

I have generated a 3D model for a GPCR using I-TASSER. how to validate the structure?

16 August 2019 2,796 14 View

I want to find out an unknown chemotactic factor that is secreted by the cells. Can any one suggest me a method of doing so?

the cells secrete some unknown chemotactic factor in media due to which they aggregate. I want to analyse and find out the unknown factor. Can any one suggest me a procedure to do that?

13 June 2019 6,802 0 View

What N terminal signal sequence can be added for expression of a membrane protein in insect cell using baculovirus expression system?

i am trying to express a membrane receptor in the insect cell system. what signal sequence should i use for expression of the receptor in the insect cell membrane?

02 September 2018 7,948 3 View

No amplification in real time PCR

I am not getting any amplification for my target gene in real time PCR. The cDNA samples are fine since the amplification in positive control is fine. I have also obtained amplification for my...

01 January 1970 2,494 3 View

Phylogenetic tree of large data

How to construct a phylogenetic tree of nearly 4000 sequences?

01 January 1970 4,560 1 View

Immunoprecipiation of G alpha

I am trying to immunoprecipitate an endogeneous G alpha subunit with its specific antibody. I have used NP40 buffer and protein concentration about 2 mg/ml but i did not obtain in protein after...

01 January 1970 1,248 0 View

Real time pcr vs conventional pcr

I am trying to check downregulation of a gene using real time pcr. Real time pcr shows that the gene haa been downregulated in my test samples and i get very less or no amplification for the gene...

01 January 1970 8,099 1 View

Anti myc confocal microscopy

I am overexpressing an endogeneous protein with c terminal myc tag in entamoeba. Overexpression has been confirmed by real time pcr. But when i am trying to visualize it in confocal microscopy...

01 January 1970 4,126 2 View

Live actin staining with sir Actin

We are trying to stain live Entamoeba cells with sir Actin( conc- 100nm to 1um) in culture medium. But we are unable to see any staining. Please suggest what may be the problem. We are also using...

01 January 1970 374 2 View

GPCR transcriptomics data

I have overexpressed a gpcr with sn unknown ligand and very less identity to known gpcrs in Entamoeba. I was thinking about proceeding for transcriptomic studies with the cell line so that to get...

01 January 1970 9,201 0 View

GPCR structure prediction and refinement

Can anyone please tell which is a good server for ab-initio modelling of GPCRs? and also a server for refinement

01 January 1970 160 2 View

Problem in cloning

I am trying to clone a 1 kb long GPCR into the vector PENTR3C which is almost 2.2 kb in length. I am trying to clone it between the KpnI and XhoI site. I am using XL1B strains. The pentr3c vector...

01 January 1970 5,021 0 View