Specific Labeling (e.g. Sel-Met) of Proteins
(Contributed by Mahavir Singh)
NOTE: SeMet is TOXIC. The culture supernatant should be collected for disposal as hazardous waste. Fill the waste bottles to shoulder-height, not all the way to the screw cap. Use the Online Tag Program to generate hazardous waste tags as soon as you start generating the hazardous waste and affix the labels to the waste bottles immediately.
Refer to your lab-specific Standard Operating Procedure (SOP) for the proper way to work with SeMet. At a minimum you will need to use the appropriate Personal Protective Equipment (PPE) which includes the use of nitrile gloves, lab coat, and full-coverage shoes.
1. Thiamine, 1% 0.5 g/50 ml (0.22um filtered)
2. Antibiotic (0.22um filtered)
3. MgsO4, 1M 13.32 g/50ml (0.22um filtered)
I ) EDTA 250 mg/50ml
II) Zn(Ac)2 420 mg/50ml
First dissolve separately and then combine (0.22um filtered)
5. Trace Elements solution
- H3BO3 2.5 g/l
- CoCl2*6H2O 2 g/l
- CuCl2*2H2O 1.13 g/l
- MnCl2.”H2O 9.8 g/l
- Na2MoO4*2H2O 2 g/l To dissolve, lower the pH with the citric acid or HCl (0.22um filtered)
6. Glucose, 20% 100 g/ 500ml (autoclave separately)
For 1 litre of medium add:
255mg Asp, Met
125mg Cytosine, Guanosine, Uracil
100mg Asn, Leu, His,
50mg Phe, Thymine, Thymidine
1g Citric Acid
1.3ml Trace element solution
36mg Ferrous citrate (dissolve in 120ul of conc. HCl)
1ml Zn-EDTA solution
Adjust the pH to 7.0
To make the final ready media, add to the above autoclaved media :
1. Glucose 25 ml
2. Thiamin 560 ul
3. Antibiotic Half of the usual amount
4. MgSO4 2 ml
5. 50mg Cys, Trp, Nicotinic acid
6. 0.1mg Biotin
Make over-day culture from freshly transformed cells in 5 ml of LB with antibiotic.
Inoculate 100ul of LB to the 100ml of Minimal Media with antibiotic.
Add 1 litre of Minimal Media to the 100ml overnight culture with antibiotic.
Induce at of 0.7- 0.8 with the IPTG (final conc. 1mM).
Grow the culture for 5 – 7 hrs. and spin it.
# Add the desired amino acid at the time of induction as well (same amount as added before, 0.22um filtered).
# For Se-met labelling use Se-Met instead of Met.
Updated: September 17, 2012Updated Monday, September 17, 2012.