Why mP values in a FP assay are decreasing?

I initially did an FP assay with receptor (A) and a flourescent labelled ATP analog (B). For the first three times, i tried varying B conc for a fixed A concentration. I found increase in mP...

03 April 2019 2,773 1 View

Co-transfection of 2 plasmids in HEK-293 cells

I am trying to co-transfect 2 plasmids in HEK-293 cells. One is pcDNA-3 based( protein A) and the other is pcDNA5/FRT/TO based GFP-tagged ( protein B). Protein A's expression is optimized. Protein...

06 July 2017 9,164 3 View

Anyone knows the IC50 values of Geldanamycin in HeLa cell line?

I am doing MTT assay on Hela cells. i keep the drug for 24 hours after cells have attached and become confluent in the 96 well format. After that i add MTT at 0.75mg/ml and incubate for 2.5 - 3...

03 April 2016 4,845 5 View

Does heat shock to Hela cells produce certain chemicals or changes in cell morphology that affect the MTT assay?

After a certain incubation period at high temperature, I am getting higher MTT readings as compared to smaller incubation periods at the same temperature. Cell viability should ideally decrease...

04 May 2015 2,371 4 View

Which cell lines shuld i use for my experiments?

The experiment i have decided to conduct is as follows: I want to overexpress luciferase in certain cancer cell - lines. After transient transfection, i then intend to overexpress Hsp90 in those...

01 February 2015 9,441 0 View

Is ATP regenerating system mandatory to use when measuring ATPase activity of a protein?

I am using a pretty old ATPase protocol that measures inorganic phosphate released by The Fiske and Subbarow method( 1925). Nothing is mentioned about ATP regenerationin the protocol. Should I add...

01 February 2015 5,295 1 View

Can inhibitors function independent of concentration.?

I am doing aggregation prevention assay of one substrate and a chaperone. the chaperone prevents aggregation upto 90% at 1:10 molar ratio. Now I add a certain inhibitor of the chaperone to see how...

09 October 2014 5,055 4 View

Use of Geldamaycin at 500nm for monitoring aggregation studies using substrate and chaperone?

Hi, i am looking to use Hsp90 Inhibitor Geldanamycin. I have optimized the assay where i have a substrate protein and this chaperone and i have monitored the prevention of aggregation using a...

01 February 2014 1,175 1 View

Why am I getting two light bands on SDS page along with my protein of interest?

I am trying to purify mammalian HSP90 from E.coli Bl21DE3 cells. They are histagged. I use Ni-NTA, followed by Anion Exchange chromatography and then GFC in Sephadex 10/300. My protein has a mol...

31 December 2013 2,683 59 View

Should I remove imidazole from protein samples before using native PAGE?

For running my samples in native PAGE, should I remove imidazole by dialysis? I have done Ni-NTA followed by size exclusion chromatography and then concentrated the samples using Amicon Filter...

03 April 2013 6,719 13 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

Kinetics of Enzyme production in Recombinant E Coli?

Hello, I am trying to find kinetic information (such as rate of enzyme production etc.) on the expression of taq polymerase in E-Coli after inserting a recombinant plasmid. Any help would be...

12 February 2021 1,386 3 View

Is it possible to quantify recombinant protein in the supernatant using SDS PAGE ?

I would like to quantify a recombinant protein (with native signal sequence) secreted by Pichia pastoris using the SDS PAGE. Since my standard protein (plant origin) is not pure, I would like to...

07 February 2021 3,880 9 View

How to measure the concentration of a labeled protein with no tryptophan or tyrosines?

Hey I'm trying to measure the concentration of a protein I've purified and then labeled with Atto 647 flourescent dye. The protein has no tryptophans or tyrosines (although does have...

04 February 2021 4,482 4 View

How to prepare protein samples in EMSA assay?

Recently l was studying EMSA assay, but l didn't find any detailed information about protein sample. we can use two kinds of protein, including crude protein extracts and recombinant proteins. but...

02 February 2021 10,047 3 View

Difference between assessing EC50 in presence of physiological levels of human serum albumin and alpha1 acid glycoprotein vs. % human serum?

01 February 2021 1,825 1 View

Alpha synuclein lack structurw (i.e some of the helix part goes into turn ), any cause behind this?

I am trying to simulate alpha synuclein (1xq8.pdb) in GROMACS using GROMOS53a6 ff. I have run it for 200 ns. At the end of simulation alpha helix protein losses its helical structure. I am not...

31 January 2021 4,285 3 View

How to increase the lower detection limit of ELISA?

Hi all, I am trying to establish an ELISA method for the quantification of a recombinant protein injected into mice. In the meantime I am trying to make calibration curves with the purified...

27 January 2021 8,702 3 View