CCP4 wiki

Expression of SeMet labeled proteins: Difference between revisions

Page Discussion

Expression of SeMet labeled proteins (edit)

Revision as of 08:23, 24 March 2010

349 bytes removed ,  24 March 2010
re-format and add non-bacterial
No edit summary
(re-format and add non-bacterial)
Line 1: Line 1:
Several methods are available for the bacterial expression of semet-labelled proteins.
= bacterial expression of semet-labelled proteins =




In the literature:
 
references:




Line 38: Line 39:
400 ml M63 stock salts
400 ml M63 stock salts


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_1: solution 1]
# 20 ml of solution 1


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_2: solution 2]
for 50 ml (in ddH<sub>2</sub>O)
add 200 mg of the following amino acids:
A, R, G, Q, H, I, L, K, P, S, T, V
pH should be adjusted to 7-7.5 with 1 M KOH
# 20 ml of solution 2


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_3: solution 3]
for 50 ml (in ddH<sub>2</sub>O)
add 200 mg of the following amino acids:
N, D, C, E
pH should be adjusted to 7-7.5 with 1 M KOH


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_4: solution 4]
# 20 ml of solution 3


20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins#solution_5: solution 5]
for 50 ml
200 mg phenylalanine
100 mg tryptophane
200 mg tyrosine
20 mg p-aminobenzoic acid
20 mg p-hydrobenzoic acid
 
dissolve in 50 ml 0.1 M KOH
 
# 20 ml of solution 4
 
for 50 ml
200 mg hypoxanthine
200 mg uracil
dissolve in 50 ml DMSO
 
# 20 ml of solution 5
 
for 500 ml (in ddH<sub>2</sub>O)
100 mg biotin
100 mg nicotinamide
10 mg riboflavin
100 mg thiamine
 
# M63 stock solution
 
for 1 liter (in ddH<sub>2</sub>O)
15 g KH<sub>2</sub>PO<sub>4</sub>
45.6 g K<sub>2</sub>HPO<sub>4</sub>
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml


2 ml of 1 M MgSO<sub>4</sub> stock
2 ml of 1 M MgSO<sub>4</sub> stock
Line 67: Line 106:




== M63 stock solution: ==
for 1 liter (in ddH<sub>2</sub>O)
15 g KH<sub>2</sub>PO<sub>4</sub>
45.6 g K<sub>2</sub>HPO<sub>4</sub>
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml
== solution 1: ==
for 50 ml (in ddH<sub>2</sub>O)
add 200 mg of the following amino acids:
A, R, G, Q, H, I, L, K, P, S, T, V
pH should be adjusted to 7-7.5 with 1 M KOH
== solution 2: ==
for 50 ml (in ddH<sub>2</sub>O)
add 200 mg of the following amino acids:
N, D, C, E
pH should be adjusted to 7-7.5 with 1 M KOH
== solution 3: ==
for 50 ml
200 mg phenylalanine
100 mg tryptophane
200 mg tyrosine
20 mg p-aminobenzoic acid
20 mg p-hydrobenzoic acid
dissolve in 50 ml 0.1 M KOH
== solution 4: ==
for 50 ml
200 mg hypoxanthine
200 mg uracil
dissolve in 50 ml DMSO
== solution 5:  ==


for 500 ml (in ddH<sub>2</sub>O)


100 mg biotin
= expression of semet-labelled proteins in other systems =
100 mg nicotinamide
* mammalian cells: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242577/pdf/2008.pdf mentions several mammalian cell lines, including CHO, HEK239, and COS. Apparently, Lec8 have been used, too.
10 mg riboflavin
* Yeast
100 mg thiamine
* stable S2 insect cells
* baculovirus expression system
Kay
Bureaucrats
1,330

edits

Retrieved from "https://wiki.uni-konstanz.de/ccp4/index.php/Special:MobileDiff/1626"