Protein Expression

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Questions related to Protein Expression

How can I confirm protein expression from Pichia pastoris colonies?

I was successful with the transformation( pPIC9K vector in Pichia pastoris GS115 strain )and got good colonies in RDB plates after 48 hours. later in MD and MM plates also. Can anyone please tell...

12 December 2019 8,902 8 View

How can I confirm protein expression from Pichia pastoris colonies?

I was successful with the transformation( pPIC9K vector in Pichia pastoris GS115 strain )and got good colonies in RDB plates after 48 hours. later in MD and MM plates also. Can anyone please tell...

10 December 2019 6,460 8 View

Is it possible for cultured bacteria that are in their stationary phase to go back into their log-phase after addition of depleted source ?

Hello all, We have a discussion in our lab and would love your input: Is it possible for cultured bacteria that are in, or approaching, their stationary phase (in our case OD 5 - 6) to go back...

10 December 2019 1,213 8 View

What is the cause of this problem in western blot?

This is the upper part of a western blot membrane, and as you can see there are some proteins that did not enter the separating gel. Does this affect the level of protein expression? I mean is...

16 November 2019 1,429 4 View

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 2,674 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 4,265 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 3,947 3 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 6,690 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 3,724 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 2,920 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 1,597 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 1,891 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 5,011 3 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 5,214 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 2,067 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 1,957 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 9,552 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 6,923 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 3,279 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 3,943 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 8,308 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 7,872 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 6,630 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 3,034 2 View

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