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2
votes
1answer
41 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
24 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
25 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 ...
1
vote
0answers
31 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
46 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 ...
2
votes
1answer
47 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 ...
0
votes
1answer
25 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
32 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
41 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 ...
1
vote
1answer
64 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
votes
1answer
54 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
votes
1answer
65 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 ...
5
votes
3answers
248 views

What are some examples of genes that code for multiple proteins?

The title pretty much says it all. It is widely taught that a gene in a eukaryotic system could produce more than one protein due to post-transcriptional modification, but I do not believe I have come ...
3
votes
1answer
110 views

What is the translation termination efficiency in mammalian cells?

When I express proteins in bacteria I put at least two stop codons at the end of the gene to increase the termination efficiency. Is this the case in eukaryotic cells too? If I put a single stop codon ...
2
votes
1answer
832 views

Where to put the gene after eukaryotic promoter for best expression levels?

As far as I know there is an optimum distance between a promoter and the gene for the best expression levels. What is that distance for common promoters like CMV, SV40? If you have a first hand ...
6
votes
1answer
241 views

What is the best way to express two proteins in a mammalian cell?

I have two proteins and I will be preparing a vector with both genes for stable transfection. Each protein will have their own promoter and I will use piggyBac vector to insert a single cassette with ...
4
votes
1answer
540 views

How useful are vitamin and trace element additions in E. coli culture medium?

In protocols for minimal media for growing bacteria I often see that a vitamin mix or a mix of various trace metals is added. But at least for E. coli I know that those are not strictly necessary, ...