Re: [ccp4bb] How to express a >95KD FAD protein

try codon optimisation use Rosetta 2 strains fuse to MBP I expressed a yeast protein of ~100 KDa, expression was low until I fused it to MBp (resulting protein ~ 150 KDa)

[ccp4bb] off topic

Hello everyone, I am trying to purify a 13 KDa membrane protein using Ni NTA. The protein is solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and binds very well to the column. However, it also turns the column brownish. The protein contains 4 cysteine residues so I suspect that th

Re: [ccp4bb] Purification

Hello everyone, Just want to say thanks for your great ideas and time to reply my question. Hope I will solve my problem soon Kien

[ccp4bb] purification

Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contamin

[ccp4bb] native gel

Dear all, I am wondering if anyone is working on Blue Natve PAGE or other alternate methods. I need some help to troubleshoot my Native PAGE experiments. It will be great if anyone can help me in this regard. I am working on membrane proteins which have pI around less than 7. i need to run Native

[ccp4bb] LDAO & SDS-PAGE

Hello everyone... I have a protein solubilised in 1% LDAO, 50 mM Phosphate pH 7.5, 150 mM NaCl. When I mix it with laemmli buffer and heat at 95C, 3 mins for SDS-PAGE, it aggregates (as in when you mix GuHCl with SDS) and can't be loaded onto the gel. Does anyone know why it happens? Thanks a lot

Re: [ccp4bb] ion exchange chromatography problem

...forgot to mention I used Q sepherose from Sigma for the IEC

[ccp4bb] membrane protein not solubilised

Hello everyone, I am working on a yeast membrane protein, about 100 KDa. The protein is expressed as an inclusion body in E coli even at 18 C. The problem is solved when MBP is fused to the N terminus of the protein. The resulting protein is ~140 KDa, pI 6.0. The expression level is moderate. Afte

[ccp4bb] ion exchange chromatography problem

Hello everyone, I have a question about ion exchange chromatography. - I have a membrane protein (pI 9.5), 13 KDa - It's fused to MBP (maltose binding protein pI 5.1, 44 KDa) to prevent inclusion body formation during high level expresscion. - The final protein has a pI of 6.1, 57 KDa --> The p