Protein Expression

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Questions related to Protein Expression

Can someone recommend a DNA sequence for a glycine/serine linker for optimal fusion protein expression in HEK293t cells?

I would like to use a glycine- serine linker to link a FLAG-tag to the N-terminus of a recombinant protein. I'm not sure which codons for glycine are optimum for expression in HEK-293t cells. Any...

11 November 2019 2,779 2 View

What's the solution of the low protein expression eficiency in yeast?

The virus protein has been expressed in Y187 strain, but no band in westernblot test. Using C-myc tag, Electrochemiluminescence Backgroud clean, The Mark and positive group goes well. Any other...

11 November 2019 4,348 3 View

KO cell confirmation by WB and IF?

Hello everyone, I knocked out a gene by CRISPR/Cas9 system, and then I confirmed it by WB, there was no protein expression, so I assumed the gene was successfully knocked out, however, when I...

11 November 2019 4,063 3 View

I want to make a gene overexpressing cell line?

I want to clone a target gene into a lentiviral vector to produce a mouse cell line that overexpresses the target gene. I want to know the difference between inserting the full-length cDNA and...

07 November 2019 4,549 2 View

Protein loading control for bacterial lysates for Western Blotting?

Hey everyone, I made bacterial cell lysates and blotted them to check virulence factors. The two virulence factors I investigated worked quite well and specific bands were visable. However, now I...

16 October 2019 6,814 3 View

How to perform Western blot analysis when the comparative untreated control does not express protein bands?

Hi, I am looking at a change in protein expression after treatment over time and comparing samples to an untreated control. However, certain proteins i.e. stress response proteins, does not...

10 October 2019 3,804 4 View

Does anyone have cellular fractionation kit preferences?

I'm looking for a nuclear extraction kit, and upon searching, every vendor offers one with each kit seemingly the same. What kit do you use, and why do you like it over the multitude of...

10 October 2019 2,993 1 View

How to improve protein expression under pBAD promoter?

I am studying the expression and toxicity of a GFP construct using pBAD24 on E.coli BW25113. First I used LB as a medium, but due to heterogeneous expression between cells I decided to use...

10 October 2019 1,690 3 View

How to isolate plasmid DNA from mammalian cells (HEK 293T) after transfection?

I have transfected the HEK 293T cells with plasmid having fluorescent protein expression. After sorting the positive cells (cells having fluorescent protein expression), i want to isolate the...

19 September 2019 2,039 3 View

How to isolate plasmid DNA from mammalian cells (HEK 293T) after transfection?

I have transfected the HEK 293T cells with plasmid having fluorescent protein expression. After sorting the positive cells (cells having fluorescent protein expression), i want to isolate the...

09 September 2019 5,106 3 View

Which strand the UCSC genome browser provides?

Hi all, I want to download a gene sequnce from Genome Browser, but I am not so sure about the direction of the sequence I get. Does it always show the coding strand sequence of one gene in the 5'...

06 September 2019 6,308 2 View

What is the best way to normalize phosphorylated protein expression?

I'm trying to analyze how the ratio of AMPKp: AMPK changes among my treatment groups. What is the best way to do this? Currently, all I've done is divide AMPKp by AMPK, and then calculate...

31 July 2019 5,331 3 View

I want to do protein expression in E. faecalis JH22 by using anhydrotetracycline (ATc) to induce protein expression .Need protocol for expression?

I want to use E. faecalis JH22 as competent cells to express my protein using anhydrotetracycline (ATc) to induce protein expression i need some standard protocol if someone has use it before.

27 July 2019 2,200 2 View

What is the best way to normalize phosphorylated protein expression?

I'm trying to analyze how the ratio of AMPKp: AMPK changes among my treatment groups. What is the best way to do this? Currently, all I've done is divide AMPKp by AMPK, and then calculate...

07 July 2019 2,077 4 View

I want to do protein expression in E. faecalis JH22 by using anhydrotetracycline (ATc) to induce protein expression .Need protocol for expression?

I want to use E. faecalis JH22 as competent cells to express my protein using anhydrotetracycline (ATc) to induce protein expression i need some standard protocol if someone has use it before.

07 July 2019 9,644 2 View

Methods to measure protein expression in plant cells?

I am looking at different methods to measure the expression of proteins in vivo in plant cells. I know that GFP-tagging is the most historically common, but I'm just brainstorming (so don't worry...

07 July 2019 7,046 6 View

What is the best choice for a ribosomal protein used as a loading control in WB?

Hi everyone. I have to assess protein expression in my adult satellite muscle cells by WB. I'd prefer not to use the conventional beta-actin or alpha-tubulin, nor GAPDH. I saw that 18S mRNA was...

25 June 2019 3,445 2 View

What AraBADC- strains do you recommend for membrane protein expression?

I am going to boldly attempt the purification of a viral membrane protein and want to try several expression systems to see what works best. One I would like to try is pBAD because (in my hands...

19 June 2019 4,104 1 View

Do I need a loading control if I'm measuring phospho and total protein expression?

I am looking to measure phospho-Erk and total-erk protein expression with western blotting, and am wondering if it is necessary to use a loading control. I normally measure phospho first, then...

12 June 2019 8,372 2 View

What AraBADC- strains do you recommend for membrane protein expression?

I am going to boldly attempt the purification of a viral membrane protein and want to try several expression systems to see what works best. One I would like to try is pBAD because (in my hands...

06 June 2019 7,951 1 View

What is the best way to solubilize an insoluble protein produced in Baculovirus expression system for his-tag purification?

Hi, I have produced a 37kda viral protein using Baculovirus expression system (FlashBac Ultra)/insect cells. It is a glycosylated protein and contains a transmembrane region at N-terminal....

06 June 2019 6,709 8 View

DE3 Bacterial cell lysis conditions in Branson SFX150 Sonicator?

Hi All, We are interested in using the Branson SFX150 sonicator we have in our lab to lyse DE3 E.coli in order to purify a couple of proteins. I have worked with larger sonicators in the past to...

29 May 2019 3,102 2 View

Can someone tell me , which vector and strain is best to express Thermomyces lanuginosus lipase in E. coli system?

Actually , i want to express Thermomyces lanuginosus lipase (TLL) in E coli system. so please help me and if somebody have protocol/procedure plz share with me. Thanks in advance!

13 May 2019 494 0 View

How would two RBSs effect expression of the fusion protein in E.coli?

If there was a very strong RBS site placed between the promoter and the AUG of the fusion partner at the N terminal, while there would also be the Shine-Dalgarno sequence placed in between the...

05 May 2019 2,547 4 View

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