Lysine Methylation: Difference between revisions
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(New page: This protocol is an NKI local adaptation based on the article by Grimes and co-workers[http://www.ncbi.nlm.nih.gov/pubmed/17098187?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Re...) |
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*1mg/ml protein | *1mg/ml protein | ||
*50mM HEPES (pH7.5) | *50mM HEPES (pH7.5) | ||
| Line 27: | Line 25: | ||
Remove precipitated protein (up to 50%) by centrifugation | Remove precipitated protein (up to 50%) by centrifugation | ||
Concentrate and put protein on Gel Filtration column in a Tris | Concentrate and put protein on Gel Filtration column in a Tris containing buffer, eg: | ||
*20mM Tris (pH7.5) | *20mM Tris (pH7.5) | ||
*200mM NaCl | *200mM NaCl | ||
*1mM DTT | *1mM DTT | ||
Latest revision as of 12:50, 18 March 2008
- 1mg/ml protein
- 50mM HEPES (pH7.5)
- 250mM NaCl
- Do NOT use Tris
Add per 1ml protein:
- 20μl 1M dimethylamine-borane complex (ABC) – freshly prepared
- 40μl 1M formaldehyde (from 37% stock)
Mix gently and incubate for 2hrs at 4°C
Add again:
- 20μl 1M dimethylamine-borane complex
- 40μl 1M formaldehyde
Continue incubation for 2hrs at 4°C
Then add
- 10μl 1M dimethylamine-borane complex (ABC)
Incubate O/N at 4°C
Remove precipitated protein (up to 50%) by centrifugation
Concentrate and put protein on Gel Filtration column in a Tris containing buffer, eg:
- 20mM Tris (pH7.5)
- 200mM NaCl
- 1mM DTT