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Questions tagged [protein-expression]

The process by which proteins, encoded in the genome, are ultimately synthesized.

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0answers
8 views

GFP intensity after proliferation and life time

I'm interested in knowing : What is the life time of GFP in a cell ? After division, does each "daughter" cell express GFP like the "mother" ? Meaning is there any loss in the expression of each cell ...
50
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4answers
8k views

What is the longest-lasting protein in a human body?

Protein life times are, on average, not particularly long, on a human life timescale. I was wondering, how old is the oldest protein in a human body? Just to clarify, I mean in terms of seconds/...
1
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1answer
27 views

Centrifuge after sonication

I follow a protocol to get protein from E.coli cells after sonication. I used to grow 6 litres of large cultures and add IPTG to express the protein. I centrifuge for 10 mins at 8,000 rpm and get the ...
2
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0answers
33 views

How are animal patterns encoded in the dna?

After seeing the patterns on the feathers of a argusianus argus pheasant (shown below), I am curious where is the information that encodes a pattern for a particular bird, and what form is this ...
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0answers
24 views

Highest yielding protein in E coli culture

Which protein can be isolated or over-expressed to the highest possible yield per liter of E coli culture. Or in other words, what is the easiest possible protein to express in a typical E coli ...
0
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0answers
18 views

Constant SDS-PAGE bands produced in the overexpression of Sf9 insect cells

We recently cloned four pFastBac1 vectors with four different target genes, respectively. All proteins have tagged with C-terminal hexahistidine tag. We followed the basic procedures to perform the ...
1
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0answers
33 views

How can I render a protein non-functional to avoid problem during expression?

We're expressing a protein in a model organism for crystallization, however it seems the protein has some toxic effect on the cells. Is there something we can do to make it non-functional during ...
0
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0answers
15 views

How can I measure the efficacy of RNAi with high sensitivity?

I'm expressing RNAi against some candidate gene using the Gal4/UAS system in Drosophila. I know protein is expressed from the gene in the targeted cell types based on immunostainings, and the good ...
0
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0answers
18 views

Will the addition of 3 amino acids in the C-terminal of a membrane protein affects its properties?

I am planning to use 2A self-cleavage peptide to express my gene of interest downstream of an intrinsic gene in my model organism so that I can exploit the intrinsic promoter, instead of cloning it ...
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0answers
39 views

Protein bands not visible after sds page coomassie dye stain

I am working with cell wall protein of Candida albicans and cryptococcus neoformans but after protein extraction and run on SDS PAGE my bands are not visible after coomassie staining. Can anyone tell ...
0
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0answers
29 views

Has network theory been successfully applied to protein expression within a cell?

I saw the following passage in the NPR 13.7 Cosmos & Culture commentary post The Big Idea Behind Big Data. Using the Internet's first generation of Big Data, researchers like physicist Albert-...
1
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1answer
455 views

Life cycle of proteins

I try to get a picture of the life cycle of a protein (considered as a specific molecule). This is how I can imagine it: After the cell is born a protein molecule is synthesized by gene expression ...
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0answers
28 views

Expressing bacterial proteins in mammalian cells

Does anyone have experience/knowledge of expressing bacterial (or other microbial) proteins in mammalian cells? I am interested in this for a project where we are trying to introduce a new enzymatic ...
3
votes
1answer
132 views

How can carnitine enter the mitochondrial matrix without OCTN2?

Some people have defects on the gene SLC22A5, giving them problems with their OCTN2 transport protein (Organic cation transport). OCTN2 transports carnitine into the mitochondial matrix where it can ...
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1answer
32 views

What are the advantages using SUMO vector for expression? [closed]

My question is : What are the advantages using SUMO vector for expression?
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1answer
401 views

Is protein production doubled if you have homozygous dominant genes as opposed to heterozygous genes?

At school we were taught in quite simplistic terms. We were told that there is a dominant and recessive type of every gene and that if the dominant gene was present, the protein is produced and if it ...
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0answers
16 views

Where I can buy an expression clone?

I need to buy an expression clone for dengue MTase to express and purify the protein. Do you know any biological company that sell them?
5
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1answer
136 views

Optimization of Signal Peptides

We are looking at modifying the signal peptide of a receptor in a common immortalized human cell line. The cell line already expresses an unusually high amount of the protein, but much of it is not ...
3
votes
1answer
296 views

why do chaperones bring protein into mitochondria? why would mitochondria need protein?

Sounds trivial? Please help to sort out. I saw this picture while looking at dehydration reactions and cell revision. And proceeded to the reference web page. Protein Folding and Processing in ...
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0answers
54 views

Protein expression in K. lactis system

I am trying to express a protein in K. lactis Protein Expression Kit (E1000S - NEB) in the way that the protein is secreted into the medium. I know that I can get some protein but it is not active. As ...
2
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0answers
25 views

Do exon deletions in C.elegans mutants necessarily result in nonsense mediated decay of the transcript?

I am trying to write out my research proposal about a project in C.elegans. There are multiple mutants of the factor that I want to study, each with different mutations. I know that a mutation that ...
0
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0answers
114 views

Problem with Rosetta gami B protein expression!

I am trying to express a disulfide rich antimicrobial peptide in the pRSET-A vector with His-tag in the Rosetta gami B DE3 pLysS bacterial host. I saw that due to its trx/gor mutations it grows slowly ...
0
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0answers
85 views

small pellet size after protein production in E. coli

I expressed a protein using 0.5 mM IPTG in E. coli (BL21 cell line straight from a glycerol stock was used as starter culture, ampicillin as antibiotic at 100 mg/L) but the cell pellets were very ...
3
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1answer
202 views

If bortezomib, a cancer drug, inhibits cell proteasomes, wouldn't resulting protein aggregate in normal cells further increase the risk of cancer?

Bortezomib is an anti-cancer drug that inhibits the proteasomes of cancer cells, allowing proteins that stop cell growth to fold and perform their function. However, wouldn't bortezomib also affect ...
3
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2answers
108 views

Examples of heterologous expression in human cell beside viral expression

Usually, people are interested in expressing human proteins in fast-growing organisms like bacteria to yield a maximum of protein within a short time lapse. Codon usage harmonization/optimization have ...
7
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2answers
309 views

Are there any examples in nature of two polypeptides joining into a single, continuous, third polypeptide?

Are there any examples in nature of two polypeptides join into a single, continuous, third polypeptide like this: (Where all the indicated amino and carboxyl groups are on the main polypeptide ...
1
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1answer
117 views

How do I improve expression using the GAL1 promoter in S. cerevisiae above the “leaky” level I'm currently seeing?

I am attempting to express GFP in S. cerevisiae using the GAL1 promoter. I always grow an uninduced (in dextrose based SD medium) culture alongside my induced (in galactose based SD medium) culture. ...
3
votes
2answers
427 views

Protein denaturation and edible toxins

As a protein denatured, it may lose its functions. So theoretically if I heat some type of biological toxins like poisonous mushroom to 100 oC or higher temperature, will I able to eat it? (I know ...
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1answer
63 views

How long ago did protein synthesis from DNA start? [closed]

How long ago did DNA start to include sequences that code for protein?
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0answers
59 views

Why does our body produce double the amount of a protein from DNA after duplication?

It is said that if a duplication mutation occurs on the DNA strand, the proteins that it is coded for would be doubled in production as a result. Why is it so? Doesn't our body have feedback ...
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2answers
1k views

Gene vs. Protein Expression Assays

Are the terms gene expression assays and protein expression assays used interchangeably in molecular biology? Or is it expected of you to differentiate between the two terms. For example, if I ...
4
votes
1answer
144 views

Reason behind using inducible promoter for heterologous expression

Protocols often seem to use regulatable promoters when expressing a heterologous gene in a host like yeast where they add something like IPTG to induce expression from (say) the lac promoter. Is ...
1
vote
1answer
223 views

How to put a gene under the control of a regulable promoter?

Suppose I have a hetrologous (eukaryotic, plant) gene sequence that I want to express in E Coli under the control of a regulable promoter (say the lac promoter induced by IPTG) how exactly does one ...
2
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3answers
991 views

T7 vs pBAD promoter strength

How do pBAD and T7 promoter strengths compare when pBAD is induced under conditions which would lead to its highest strength? Are there any papers that compare the two inducible promoter systems?
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1answer
32 views

Methods for varying insert size in E. coli clones

I'm writing my master's thesis proposal, and I need to improve the solution-behavior of some truncated protein domains which have previously shown poor solubility when expressed in E. coli. To ...
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0answers
34 views

Computer Models of E Coli / Saccharomyces cerevisiae metabolite pathways

Are there mathematical models one can use to predict the expression levels of a foreign gene inserted into a well studied host organism like E Coli or S cerevisiae? I'm not, obviously, looking for ...
4
votes
0answers
248 views

Relative densitometry from SDS PAGE

I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software. My questions are ...
2
votes
2answers
135 views

Are multi-chain proteins synthesized as one biological unit?

Are multi-chain proteins (especially homo/hetero-dimers) synthesized together as one overall unit or are they separate monomers which bind together at some point after synthesis, and are there any ...
3
votes
1answer
2k views

How is the subunit molecular weight different from the native molecular weight?

I noticed that the native molecular weight for an enzyme is different from its subunit molecular weight. Why are they different? Aren't the genes needed to express the enzyme the same in the native ...
2
votes
1answer
70 views

Do I have to use sucrose to induce a lac promoter?

I'd like to optimize the expression of a Fab fragment in Escherichia coli. For induction of the lac promoter on the pAK400 vector I use IPTG and sucrose. Do I optimize the expression in case I would ...
4
votes
1answer
146 views

Is it possible to express the cistrons from a polycistronic insertion fragment in a single plasmid?

I have a insertion fragment that I wish to express from pUC19 in Escherichia coli. The insertion fragment is a sub-section from a larger operon sequence and contains just the last two cistrons from ...
2
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0answers
141 views

Why HIS tag is not detected in WB or dot blot?

I would like to know why the recombinant protein with his tag at c terminus is not being detected neither in western blot or dot blot. I'm trying to validate protein expression of four proteins. ...
2
votes
0answers
116 views

Which method would more accurately help to identify the unknown concentration of a protein sample between the A280 and the Bradford methods?

I quantified my protein using the standard Bradford method and the A280 methods and obtained values that were far off from the theoretical value of the protein of interest, and therefore was wondering ...
3
votes
1answer
511 views

Western blot alternatives

Given an in vivo sample from an experimental infection, I would like to see if a bacterial protein is present. I've been thinking about Western Blot using sera from animals infected with the bacteria....
0
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1answer
42 views

If gene differs between chromosome pair does individual get a mixture of protein shapes?

Do diploids such as humans end up with a mixture of two different shaped proteins where the appropriate gene is expressed differently in the two halves of a chromosome pair or is there some governing ...
5
votes
1answer
37 views

Need to make a library of mammalian coding sequences

I want to clone ~100 different coding sequences from human or mouse into an expression vector to analyze phenotypes in tissue culture. I can do all of the steps, but I'm not sure what I should use as ...
5
votes
1answer
68 views

What's the duration between the binding of adrenaline to β-AR and the first translated protein?

Binding of adrenaline (epinephrine) to the β-Adrenergic receptor leads to formation of cAMP (via G protein activation), activation of protein kinase A and subsequently to the expression of specific ...
2
votes
1answer
174 views

can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?

Dear fellow biochemists, I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
2
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1answer
76 views

Epistasis when interacting loci are codons within a single gene

There is epistasis when the effect on the phenotype of one gene is influenced by one or more other genes (called modifiers). Is there a similar concept when the effect on the phenotype of one site ...
0
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1answer
90 views

bioinformatics /genomics [closed]

i am looking for likely truncation sites to prove recombination in sequences. i have 4 sequences. i believe one to be the "real sequence" and the other 3 to be recombinants caused by cleavage possibly ...