05 May 2013 1 5K Report

I want to block HEK293T cells in the G2/M phase after transfecting them with the construct of my interest via a double thymidine/nocodazole block. Does anyone have a protocol or suggestion regarding this?

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Databases for protein?

Hello All. I have been working on a set of proteins related to cancer initiation and progression. I wish to know the databases wish may provide me the information for the status of any protein...

01 February 2016 5,838 6 View

ShRNA mediated knockdown showing effects in the protein level but not in the RNA level, what might be the reason?

Hello everyone,  I have been trying to standardize the knockdown of my gene of interest with the shRNA bought from DHARMACON, which targets the coding sequences. Though I get reproducible results...

04 May 2015 5,609 7 View

What is the approximate time for mammalian cells that have been synchronized for a particular phase to become asynchronous again?

Does it depend on the phase in which it has been blocked, i.e. G0/G1 will take one time while G2/M will have another? Does it depend on the cell's type (dividing cells) or the cells lines one is...

04 May 2013 9,521 3 View

Flag tagged clone not expressing.

I had cloned my gene of interest (GOI) into pCMV10-3X-Flag vector for expressing into mammalian cells. Even though the gene has been cloned and sequence confirmed, I am having a problem with...

04 May 2013 4,556 10 View

Protein binding too tightly to the beads.

I have been trying to elute protein of my interest tagged to GST using a protocol containing 10mM reduced glutathione buffered at pH 8.0. However, contrary to the proposed protocol for this...

04 May 2013 352 10 View

Finding new interacting partners for my protein of interest (POI).

I want to find new interacting partners for my protein of interest(POI), but I do not want to follow the more conventional and time taking methods like the yeast two hybrid system which also may...

31 December 2012 1,202 9 View

What causes a protein to get degraded or loose its activity if stored for a long period at 4 degrees?

I had purified a kinase of my interest to near homogeneity and had stored it at 4 degrees for approximately a month to carry out assays with it. However, I found the kinase activity to have been...

31 December 2012 6,217 11 View

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