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visits member for 1 year, 8 months
seen Jul 30 at 23:14

Jul
2
awarded  Curious
Dec
13
awarded  Yearling
Apr
15
revised Tool for nucleotide alignment with all nucleotide codes (e.g. R, Y, W, S, etc.)?
added a resource
Apr
15
asked Tool for nucleotide alignment with all nucleotide codes (e.g. R, Y, W, S, etc.)?
Apr
10
awarded  Commentator
Apr
10
comment What is the translation termination efficiency in mammalian cells?
This is a synthetic gene. I already designed a primer with 3 stop codons at the end of the gene. I will post here again if I see any improvement in gene expression (with compared to other similar constructs in our lab).
Apr
10
comment Where to put the gene after eukaryotic promoter for best expression levels?
Thank you all for your comments. I did look to other common constructs and decided to leave around 40 bp between promoter and the initiation codon (this was the minimum distance I could leave because of the restriction site limitation). @Bitwise, I am using mammalian cells (specifically; fibroblast and MDCK cells).
Apr
9
asked What is the translation termination efficiency in mammalian cells?
Apr
9
comment What is the best way to express two proteins in a mammalian cell?
Thanks for your reply. I looked into the bidirectional promoters too but decided to try two different promoters. I really didn't want to go with IRES or 2A because I am too afraid to interfere with the interaction, better not risk it :). I will start the experiment shortly and share my experience in here when I get some results.
Apr
9
asked Where to put the gene after eukaryotic promoter for best expression levels?
Mar
28
revised What is the best way to express two proteins in a mammalian cell?
a typo has been corrected and grammar imporved
Mar
27
asked What is the best way to express two proteins in a mammalian cell?
Feb
9
comment Why is absorbance at 280 nm for protein solution going up when I measure repeatedly?
I diluted the protein with the same buffer I it was in. Sorry for the confusion. Thank you for the paper.
Feb
8
accepted Why is absorbance at 280 nm for protein solution going up when I measure repeatedly?
Feb
8
comment How to wash the column during protein purification with GST tag?
Bradford idea is really good actually, I will try that in my next purification. Thank you!
Feb
8
asked How to wash the column during protein purification with GST tag?
Feb
8
awarded  Supporter
Feb
7
comment Why is absorbance at 280 nm for protein solution going up when I measure repeatedly?
These are really great suggestions but most of the time I don't have much protein. I tried using higher volumes but I don't get much absorbance; they are very close to background absorbance. If I use too much protein I both waste my samples and a huge meniscus forms which prevents me from determining the pathlength. I always pipette them very well to make sure they are well mixed too. But your suggestion for measuring for an hour and plotting a graph is a good idea, maybe UV lamp is too old. Thank you!
Feb
7
comment Why is absorbance at 280 nm for protein solution going up when I measure repeatedly?
It is in phosphate buffer in stock solution.
Feb
7
comment Why is absorbance at 280 nm for protein solution going up when I measure repeatedly?
I don't have any precipitation in stock solutions and I think it wouldn't precipitate after I dilute them.