Sudden stop in recombinant protein expression in BL21?

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Peptide lost during reverse phase HPLC?

Hello; I've dissolved my synthetic peptide in pure acetic acid and tried to purify it with reverse phase HPLC. I got weak peaks during analysis, however I injected at least 5 mg of my peptide!...

02 March 2019 9,491 5 View

How to calculate Kd with flowcytometric analysis?

Hello; I want to calculate Kd value for a FITC-conjugated peptide which specifically binds to a cell receptor. I've studied some protocols, but still confused! Does anyone have any experience or...

11 December 2018 7,119 2 View

Any protocol for solution based ELISA?

Hello; I've isolated peptides against PDL-1 from a phage library, but I can't confirm their binding to target in a solid based ELISA assay. So I want to check whether if I can get a positive...

04 May 2018 1,971 3 View

What is the easiest way to remove SDS following protein purification from gel?

I've purified my recombinant protein from SDS polyacrylamide gel and now I need to remove SDS for further work. Is it possible to do a buffer exchange on amicon filters? could anyone suggest any...

01 February 2017 1,165 4 View

Does anyone know how to justify the sample size in animal studies?

Hi there; I want to investigate anti tumor effects of an antagonist in murine models and I need to know how to determine number of animals in each group such that the results would be...

11 December 2016 1,166 9 View

What does the last number in cell line name mean?

Hi there; Does anyone one know what's the difference for example between SK-MEL-28 and SK-MEL-37? what does that number mean?

11 December 2016 3,576 0 View

Is there any way to induce tumorogenesis in murine models using human cell lines?

Hi there; I need to evaluate an anti cancer vaccine against a human protein in murine models.So I should use human cell lines. Is there any convenient way to induce tumorogenesis in murine models...

09 October 2016 1,859 3 View

Is there any convinient assay for in vivo gene expression evaluation?

Hi there; I want to needle inject Balb/C mouse with a plasmid coding for a specific gene and also GFP as a reporter and I need a method for in vivo gene expression assay! Is there any special kit...

06 July 2016 3,577 3 View

Why it's optimal for industrial microorganisms to grow in high temperature?

Hi there; I've read somewhere that growing in high temperatures is an advantage for industrial microorganisms; but unfortunately I couldn't find any reason for this in related articles! Is there...

04 May 2016 3,022 5 View

How can I detect secretary GFP-fused proteins in DMEM supernatant?

I've transfected HEK293-T cells with plasmid expressing for a secretary GFP-fused protein. I would like to know is there any method rather than ELISA test to detect my protein in supernatant using...

04 May 2015 4,653 4 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 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

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

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

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

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

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View