Eleanor Martin

11 Questions 17 Answers 0 Followers

Questions related from Eleanor Martin

Can overlap extension PCR be used to simultaneously insert and remove base pairs?

I want to use overlap extension PCR to insert a domain into my construct. However I have just realised that upstream of the region where I want to insert the domain there are 19 amino acids...

23 May 2017 5,342 4 View

Can anyone advise on now to compare ligand binding sites in NMR and crystal structures?

New to crystallography...I want to compare ligand binding in a crystal structure to one generated using NMR. I have tried the pdbsum and the pisa servers but the hydrogen bonds that are identified...

28 December 2016 1,669 3 View

Does any one have any experience of creating a stable cell line using Calu-3 cells?

I am looking to create a stable cell line using Calu-3 cells. My initial plan is to co-transfect with pBABE-puro and select with puromycin. Does anyone have any similar experience with Calu-3? I...

17 August 2016 3,535 5 View

Do I need to codon optimise my sequence for expression in a mammalian cell line?

My POI is a human protein. However we when we had the DNA sequence synthesized we codon optimised for S.cerevisae (for protein expression). However, I now wish to do some interactome studies in a...

21 July 2016 1,658 3 View

When is it appropriate to use the 'sequential binding sites' model when analyzing ITC data?

I am new to ITC. I am currently using the technique to assess binding between a protein and a peptide to which it binds. The protein has two binding sites for the peptide, however site two is...

14 June 2016 9,609 4 View

What's the difference between the AKTA prime and AKTA prime plus system?

Hello all, I want to fix a Superose 6 10/300 GL column to our AKTA prime system. However, the product info says that the Superose 6 10/300 GL is not suitable for use with the AKTA prime plus...

10 March 2016 7,499 5 View

How do I determine the protein concentration of a peptide?

I am working with a construct containing only the C-terminal region of my POI. It is 42 residues long, and contains no aromatic residues (but does contain a number of basic amino acids). Following...

16 November 2015 4,131 4 View

Can anyone give me some advice on coexpression in yeast?

Hello, I would like to co-express a large (250 kDa) chloride ion channel, along with one of its interacting proteins (50kDa, soluble) in yeast. We have already optimized the expression of the...

15 October 2015 3,831 3 View

Can anyone recommend a protocol for plasmid extraction from yeast which yields DNA suitable for DNA sequencing?

The gene I am working with is toxic to bacteria and frequently causes random deletions/insertions when cloning in E.coli. Therefore I have cloned my construct directly into yeast (my expression...

07 August 2015 657 9 View

Can anyone give me some advice on native PAGE?

I want to run a native PAGE gel of my purified protein as we suspect that it may be forming oligomers. The protein I am working with is a 50kDa soluble protein with an acidic pI. I recently ran a...

26 May 2015 8,504 11 View

Any suggestions to prevent plasmid rearrangement following transformation?

I am trying to remove the GFP tag from our construct using the NEB Q5 SDM kit. My primers are designed to flank the region to be deleted and when I look at my PCR products on a gel (before the...

23 April 2015 7,449 5 View