Protein Expression

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Questions related to Protein Expression

How to induce AOX1 promoter in Pichia pastoris on agar?

I could not really find a protocol specifically stating how to induce protein expression under AOX1 promoter in Pichia pastoris. Do we straight away streak them out on BMMY agar? Or streak them...

16 March 2020 9,844 3 View

Which is the optimal optical density for toxical recombinant protein expression in E.coli BL21 (AI)?

Hi everyone. I am trying to express and purify a recombinant antimicrobial peptide using as a host cell E. coli BL21 (AI) (employing L-arabinose and IPTG). Because this peptide can be toxic to...

20 February 2020 5,070 4 View

Inconsistency in Adenoviral infection in breast cancer cell line (MDA-MB-231)?

I'm trying to inhibit a signalling pathway in a breast cancer cell line (MDA-MB-231) through overexpressing its physiologic antagonist via adenoviral infection. The problem I'm facing is the...

14 February 2020 3,513 0 View

Alternative Media for Expi293s (Currently Using F17)?

Does anyone know of a more affordable alternative to F17 media for 293Expis? We currently supplement with L-glutamine and Pluronic F-68.

11 February 2020 766 1 View

Is there a variation in CMV expression in between individual cells after lentiviral transductions?

In line with previous data suggesting low efficacy of the CMV promoter in some cell lines (Qin et al. PlosOne 2010), I experience low transcriptional activity in a neuroblastoma cell line after...

11 February 2020 2,332 3 View

Which is the optimal optical density for toxical recombinant protein expression in E.coli BL21 (AI)?

Hi everyone. I am trying to express and purify a recombinant antimicrobial peptide using as a host cell E. coli BL21 (AI) (employing L-arabinose and IPTG). Because this peptide can be toxic to...

02 February 2020 5,954 4 View

Phosphorylation of kinase domain in bacterial expression system?

Hello. I want to crystallize kinase domain of a protein in active form. The active form of the domain has specifically phosphorylated tyrosine residues. However, I am not sure if I can do so in a...

02 February 2020 7,563 5 View

Why is cell-free protein sythesis done less frequently than traditional protein synthesis?

It seems to me that cell-free protein synthesis is not very used when it comes to protein expression, though its usage should avoid many problems, like the presence of inclusion bodies. I was...

02 February 2020 7,668 3 View

How to design a plasmid for stable transfection into HeLa cell?

Hi everyone, I am a newbie in molecular biology. Recently, I encounter an issue that the non-uniform fluorescence protein expression level in lipofection transfected HeLa cell. The variation would...

02 January 2020 7,253 4 View

Hello. Can anyone recommend a hand blender that can be used to homogeneize meat samples for microbiological analysis?

I'm implementing a small microbiology lab and requiere an homogenizer for the meat samples we produce. A small hand blender will sufice, since the amount of yearly samples will be small, hence a...

02 January 2020 1,529 1 View

Please let me know, what is difference between BL21 (DE3) and BL21 (CDE3)?

Hello Experts, I am planning to do the protein expression in bacteria. but my lab has only BL21 (CDE3). I have been searched a lot, but I couldn't find any information. Can you tell me the...

01 January 2020 1,395 3 View

Is it possible for more than 1 type of plasmid enter the Pichia pastoris?

I have a mixture of plasmids (with Origin of replication that can work in Pichia pastoris) with different genes for protein expression. Is it possible that after chemical heat shock...

22 December 2019 5,737 3 View

Why our truncated protein plasmid expressed correct length of mRNA but incorrect length of protein?

Our truncated protein expressions (labeled by the HA tag) in lane 4 and 5 were supposed to be matched with the mRNA expressions in lane of 293FT-4 and 293FT-5. However, the different truncated...

20 December 2019 3,399 7 View

Why our truncated protein plasmid expressed correct length of mRNA but incorrect length of protein?

Our truncated protein expressions (labeled by the HA tag) in lane 4 and 5 were supposed to be matched with the mRNA expressions in lane of 293FT-4 and 293FT-5. However, the different truncated...

12 December 2019 5,402 7 View

How can I confirm my protein expression in MCF-7 spheroid cell through western blot ?

Dear all, I checked my protein expression in the sphere cells through western blot. I could not see any expression. I added whole cell lysate and sphere cells. I could not see any expression of my...

12 December 2019 8,543 3 View

Protein expression level read out from WB is incompatible to IF?

Hey, I did WB and IF in different time but with the same cell line, however, the results from WB and IF seem incompatible. WB and IF both show the protein expression level right? How could they be...

12 December 2019 6,504 4 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...

12 December 2019 8,940 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 6,521 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 1,270 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 1,455 4 View

What is the best way to transfer the spore suspension stocks of Streptomyces sp. to the production medium (MSM)?

I need to grow my bacteria which is Streptomyces sp. from spore suspension method. I have grown them onto the nutrient agar by spread plate method and now I incubate them for 5 days. And for the...

15 November 2019 1,169 3 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 2,709 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 4,292 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 3,992 3 View

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