Can anyone help with brain tissue protein solubilization in Lammelli buffer after precipitation?

I had homogenized protein in RIPA buffer but was facing problem in running SDS-PAGE gel. So I precipitated protein with 100% TCA , washed with Acetone and then tried to resolubilize protein in 2X...

03 April 2015 5,589 3 View

How to prepare cuprizone pellets?

What can be used to bind the powder to prepare pellets?

07 August 2014 1,938 3 View

Query regarding small size protein transfer on Nitrocellulose membrane ( pore size- 0.45u).

My protein is of molecular weight below 20 kDa. I transferred it for 30 min at 90V but could not see distinct bands i.e. smear type on Ponceau staining.15% gel was used and was run half of the...

11 December 2013 9,284 3 View

Related to western blot protein bands on X-ray film?

Transfer of proteins on nitrocellulose membrane is fine. I am following the standard protocol but I am not getting band of b-actin on X-ray film so can anyone suggest what could be the problem?...

08 September 2013 4,674 3 View

Is there any reliable bioinformatics tool for promoter sequence retrieval and primer designing for the same?

for CHip analysis

11 December 2012 6,391 7 View

do the E3 ligases which are present in stem cell stays /expresses in differentiated cell or they are regulated by specific protein expression?

topic: protein degradation by Ubiquitination for each of the protein degraded by ubiquitination , cells have specific E3 ligases. this E3 ligases are regulated by degradation of them by their own...

31 December 2011 4,109 4 View

No title

02 March 2021 3,060 3 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 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

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

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 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