Cibacron Blue versus Heparin for DNA-binding protein purification. Any advantages of one over the other?

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Are there any other "innocuous" proteins (other than BSA) that can be added to an assay buffer conditions to stabilize an enzyme?

We are working with a reductase enzyme that is very unstable at dilute concentrations (functional dimer, likely dissociating when dilute). We've explored increasing salt and buffer...

05 June 2018 6,555 2 View

How to extend the activity of a dimer enzyme in a biochemical asay?

The enzyme is a functional dimer that is losing activity rapidly when diluted to low concentrations (e.g. 50 nM). We believe this is due to dissociation. We have optimized the buffer and pH...

31 December 2017 464 3 View

Can use of a His8-tag (or His10) vs a His6-tag improve protein solubility in difficult cases? If so, to what extent?

Attempting to improve a target protein's solubility in recombinant expression. Currently His6 tagged and experiencing both low yield (inclusion bodies), and poorly soluble product (difficulty...

07 August 2013 3,650 4 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 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

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 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

Rogerio R Sotelo-Mundo

Besides heparin, hydroxyapatite may be a good choice to try. I guess that is a natural protein (not recombinant) son His-tag is not an option.

Shivakumara Bheemanaik

Heparin is a good option. This works well for DNA binding proteins.

Sunil Kumar

I have used a library of 96 cibacron blue based affinity dyes. I guess your target DNA may bind with one of the dye. These psedo affinity dyes and you need to screen first. Let me know if you need the full text or if you have any other query.Article Discriminatory protein binding by a library of 96 new affini...