Can anyone help with measuring protein concentration after PEG conjugation?

I've conjugated a PEG4-maleimide (MW 613.66) onto an antibody fragment (52 kDa), but I'm trying to figure out how this affects A260/280 measurements on a Nanodrop afterwards. After conjugation and...

04 May 2014 7,664 7 View

Affects of imidazole on protein solubility and labeling reactions?

I have purified a protein from a Ni-NTA column and noticed that during dialysis (same buffer used for elution, without imidazole) the protein precipitates. I've spun down the precipitant, and I...

08 September 2013 3,561 18 View

How does one decide on buffer choice for protein solubility?

I have a protein that I've purified from a His column. I've noticed that when I do a buffer exchange of the eluted fractions into PBS (pH 7.3) and then concentrate the fractions, the solution gets...

06 July 2013 8,821 25 View

Addition of copper sulfate to cell culture media

I'm trying to express an antibody fragment that is covalently held together by cysteine residues that I have added to the sequence. After running the western (non-reduced) to assess expression, I...

03 April 2013 470 7 View

Codon optimization of mammalian genes

I am having a gene synthesized (~1300bp) that was originally expressed in E.coli but I want to use it in a mammalian expression vector. I designed the gene and had it optimized by GeneArt from...

01 February 2013 2,914 1 View

Stable cell line creation?

I'm trying to create a stable cell line of NSO cells that express my protein into the supernatant. I've transfected the cells, seeded them at various densities into 96 well plates and screened the...

11 December 2012 6,206 6 View

Elution from 6His column

I have a 6his-tagged protein that I am secreting into the media and eluting from the Ni-NTA column, but yields seem to be really low (recovered ~300ug/L of media). I've tried running a gradient...

07 August 2012 8,248 25 View

Does anyone have experience with expressing antibody fragments using the pEE12 system in NSO cells?

I've been using a lentiviral-based system with a pLenti expression vector in 293T cells and can purify the fragment, but yields are excruciatingly low (~300ug/L). There is quite a bit in the...

06 July 2012 3,139 1 View

Protein purification from culture supernatant

Hello protein gurus, I'm expressing a protein (~50 kDa) that contains a 6x-His tag from 293T cells into the media supernatant The media is complete DMEM (10% FBS, 1% PSG, and 0.1% Blasticidin)....

05 June 2012 3,142 44 View

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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

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

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

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

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28 February 2021 9,936 2 View

When is it unnecessary to change organisational culture?

As a former management practitioner, I recall a generally accepted view that changing organisational culture is difficult, so you should only attempt it if the organisational change that you are...

27 February 2021 2,595 8 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View