Why stop the reaction in ice for the fluorescent probe FDA (fluorescein diacetate)?

Hi, In literature I noticed that who stains haemocytes with FDA for 30 min then stop the reaction in ice while this does not apply for 15 min incubation time (or maybe authors did not write this...

08 February 2021 3,366 3 View

Which is the importance of fixing (e.g. bivalvia) haemocytes and use anti-clotting solutions in flow cytometry?

I am trying to optimize a flow cytometry method to characterize mytilus spp. haemocytes. In literature I found that someone, to avoid clotting, use anti-clotting solutions or dilute and fix the...

05 November 2020 2,522 4 View

Has any of you encountered issues with protein production in a E.coli cell-free protein expression system?

I'm having trouble synthesizing a mollusk 173 aminoacids protein of unknown function in a E.coli cell-free protein expression system. The positive control works well and the plasmid has been...

01 October 2019 8,829 7 View

How I could set up a lipoplexe-based transfection protocol and compare data with commercial Viromer Red transfection reagent?

I am new in the field of transfection and I'm having a lot of problems in setting up the protocol with LIPOPLEXES in order to compare data previously obtained using Viromer Red protocol on THP-1...

13 November 2018 3,545 1 View

Does anybody know a reasonable (in prize) company where to buy an environmental chamber to adapt a confocal microscope for live cell imaging?

We are interested in adapting a Confocal microscope for long-term live imaging, and for this reason we are trying to get a small environmental chamber at a reasonable prize. It could be enough...

16 May 2017 5,437 2 View

How to store a purified protein before a kinase assay?

23 September 2013 8,697 1 View

Is it possible to position the HA taq not at the extreme end of a protein and visualize the taq-protein by Immunocytochemistry?

06 June 2013 5,942 9 View

What are the best antibody markers to detect and quantify the circulating endothelial cells and progenitors by flow cytometry?

CD45/CD31/CD34/KDR or CD45/CD31/CD146/CD133? Or both or neither?

18 February 2013 1,958 2 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

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 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

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

What urea buffer can i use for biotagged protein extraction from streptavidin beads?

Hi all, I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by...

24 February 2021 5,749 3 View

Can I use a protein-devoid condition as my negative control in ELISA?

Hello, I am running a sandwich-type ELISA where I am trying to inhibit a protein-protein interaction using small molecules. For each experiment, I determine my Z' (Z-prime) to determine the...

23 February 2021 7,429 1 View

Is there a way to extract total RNA and protein from cell culture at the same time, without denaturing protein?

Hi all, Is there a way to extract RNA and protein from the same cell culture sample? Because we need to use the protein for gelatin zymography at later time, we are looking for a method that can...

22 February 2021 5,341 5 View