Rafael Brandão

10 Questions 12 Answers 0 Followers

Questions related from Rafael Brandão

Why do my NIH 3T3 cells die after single cell sorting?

I have been sorting NIH 3T3 based on GFP positivity for quite a long time and this has worked fine when all the GFP positive cells are sorted to the same well (of a 24 well or 12 well plate). The...

06 June 2014 2,348 10 View

Use of ELISA for protein-protein interaction detection

I'm trying to optimize an ELISA-based technique where I coat with a protein, block, add a second protein and then detect for the second protein. Do you know any effective protocol? Also, how...

03 March 2013 7,032 7 View

GST interaction with other tags

Do you know (from your experience of literature), if GST (tag) interact with other tags? For example HA, HIS, MBP, etc.

02 February 2013 4,180 3 View

How fast should an ELISA test be?

I'm running several ELISA tests at the moment and it seems that sometimes they work properly and sometimes they don't. I coat overnight (4ºC), then the next day I block and incubate with primary...

01 January 2013 474 9 View

ELISA coating buffer for acid protein

I´m trying to coat an ELISA plate with a protein which pI is ~5.20. I´ve been using a bicarbonate/carbonate buffer for the coating process, however it seems that my protein does not attach to the...

01 January 2013 7,206 10 View

Can I re-incubate ELISA plates with primary Ab?

I'm running some ELISA experiment plates coated with an extremely expensive protein. However, at the end of the experiment there was no signal. The last step of this experiment was the incubation...

01 January 2013 7,768 7 View

Protein precipitates after GST purification, how to avoid this?

I´ve been trying for a long time to express a cytoplasmic tail of a transmembrane protein and purify it. Since the beginning I have experienced several problems with inclusion bodies. I was able o...

10 October 2012 4,355 11 View

Will an His tag decrease solubility of a human protein expressed in E. coli?

I have already tried with GST but got some inclusion bodies issue and then I thought His tag would be more suitable. However, I have been reading about it and it seems that in many cases it can...

10 October 2012 8,558 8 View

How can I get my GST fusion protein to bind Sepharose 4 Fast beads in the presence of 0,1% Sarkosyl?

I´ve been trying for a long time to solubilize my protein using Sarkosyl, which was expressed as inclusion bodies. I have incubated my pellet in Sarkosyl 2% in order to solubilize and then...

10 October 2012 3,699 13 View

Solubilization of proteins from inclusion bodies: 6M Guanidine or 8M Urea?

I´ve been trying to solubilize proteins from E. coli inclusion bodies using several methods. The last one that I´ve tried was 6M Guanidine. However it seems that it did not help. I would like if I...

10 October 2012 9,330 16 View