1,330
edits
Expression of SeMet labeled proteins: Difference between revisions
Jump to navigation
Jump to search
re-format and add non-bacterial
Jacobkeller (talk | contribs) No edit summary |
(re-format and add non-bacterial) |
||
| Line 1: | Line 1: | ||
= bacterial expression of semet-labelled proteins = | |||
references: | |||
| Line 38: | Line 39: | ||
400 ml M63 stock salts | 400 ml M63 stock salts | ||
20 ml of | # 20 ml of 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 | |||
# 20 ml of 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 | |||
20 ml of | # 20 ml of solution 3 | ||
20 ml of | 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: | ||
= expression of semet-labelled proteins in other systems = | |||
* 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. | |||
* Yeast | |||
* stable S2 insect cells | |||
* baculovirus expression system | |||