Ashish Kumar

13 Questions 6 Answers 0 Followers

Questions related from Ashish Kumar

Working protocol on Taxol-washout for microtubule assays ?

Hi, Can anyone please give a working protocol for Taxol washout experiment, I am trying to study the impact of MAPs on Microtubule stability. I am using flow-chamber, attaching Taxol (1uM)...

02 July 2020 890 0 View

What is the catastrophe frequency during microtubule polymerization?

I am trying microtubule polymerization in-vitro for TIRF analysis. I am using the standard protocols published from different labs. A) I have an observation that catastrophe is very rare in my...

16 December 2019 6,422 3 View

BL21(DE3) pLysS cells ?

I recently transformed BL21(DE3) pLysS cells (same batch) with three different plasmids two pET30a and one pGEX6P2, separately. As we know, that BL21(DE3) pLysS cells are resistant to...

31 May 2019 629 4 View

How do you measure microtubule polymerization and depolymerization velocities, catastrophe and rescue frequencies ?

Hello Everyone, Can you suggest me on how to quantify microtubule polymerization and depolymerization velocities, catastrophe and rescue frequencies using TIRF. Thank you.

10 May 2019 5,808 0 View

Fluorophore proteins (eg GFP, YFP, RFP) expressed in bacterial expression system are active?

Hi everyone, I have to express different mammalian proteins tagged with different fluorophores (such as GFP, YFP and others) under bacterial expression system and to use these purified proteins...

25 April 2019 1,623 3 View

Is DsRed tag is suitable for bleaching experiments?

Hello, I have been doing FRAP on GFP tagged proteins using CLSM510META without any problem, but recently tried bleaching DsRed (monomer) tagged proteins. I used HeNe laser (543 nm) up to 100%...

01 September 2015 1,490 1 View

How pure is Ribosome purification from cultured cells; without any other cytoplasmic contaminations?

I followed the available protocols, link given, to purify cytoplasmic ribosomes from HeLa cells. After the ultracentrifugation, ribosome pellet was washed briefly 3-4 times. However, I can see the...

08 August 2015 3,226 3 View

Do I need to include plate-bound/soluble anti-human CD3/CD28 and rhIL-2 in PBMC culture for Treg stimulation?

I want to study the recall response of human regulatory T cells to some viral antigens. Following is the outline of my experiment. Isolate PBMCs from individuals who have been previously exposed...

23 June 2015 3,343 7 View

Do we have some specific cytoplasmic retention tags to keep nuclear proteins away from the nucleus?

Is there some way to restrict a nuclear protein from going to the nucleus. I do not want to mutate/delete the NLS, rather would prefer to tag my protein to some cytoplasmic retention tag. Could...

16 February 2015 817 7 View

Is anyone familiar with BrdU / BrUTP incorporation?

I have a query on BrdU/ BrUTP incorporation in culture cells. Why is transfection/microenjection required for BrUTP incorporation. Whereas, We add BrdU directly into the culture medium to pulse...

09 October 2014 3,703 6 View

Cell cycle..

hi everyone...i have to see the effect of certain chemical compound on the cell cycle..basically i have to see their effect on the check points,whether it is causing cell cycle arrest at any...

28 September 2011 5,247 1 View

Microtubule polymerization and depolymerization velocities, catastrophe and rescue frequencies

Hello Everyone, Can you suggest me on how to quantify microtubule polymerization and depolymerization velocities, catastrophe and rescue frequencies using TIRF. Thank you.

01 January 1970 5,152 2 View

Problems of microtubule dynamics experiment with TIRF_photobleaching, stability, etc

We are trying to study microtubules dynamics using TIRF. The labels used for tubulin labeling are Hilyte-488, Alexa-568 or Atto-565. We are seeing very rapid photobleaching within seconds of...

01 January 1970 3,765 2 View