What it is better to amplify 3'UTR from genomic sequences of by RACE using RNAs?

11 December 2016 251 0 View

What is the best and easy software to analyze promoters?

I used genomatyx to analyze transcription factors in promoters sequences, but now I have many problems to login, so does anyone knows another facility to do this?

09 October 2016 5,956 5 View

Too much beta lactamase in protein expression BL21 cultures?

06 July 2016 9,412 5 View

Why is recombinant mutant protein expressed less than wild-type?

09 October 2015 1,153 7 View

Bad resolved band in EMSA?

Sometimes it is not possible to see single bands in EMSA, and complexes remain as a big spot not resolved. Could anyone explain to me how to find a solution?

09 October 2013 3,564 5 View

Bands in EMSA gel remain as a big spot, not well resolved, is it impossible to see single bands?

Could anyone bring me a solution?

09 October 2013 1,197 0 View

Protein is not visible on western blot of transfected cells by confocal microscopy

I made cDNA constructions to produce proteins with GFP or dsRED (clontech), and one construction with dsRED after transfection in HeLa cells. I obtained RNA by qPCR, red cells by confocal...

31 December 2012 866 14 View

How can I increase release of my GST-fused protein after digestion with Prescission?

I bind my fused GST-protein to glutathion beads and I cut with Prescission at pH 7.5 to eluate overnight in batch method, sometimes my protein (always the same clone) remains attached to beads...

01 January 1970 6,346 5 View

No title

02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View