LOCAL

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.

 

Stock solutions

 

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)

4. Zn-EDTA:

      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:

 

400mg                       Ala, Gln, Glu, Arg, Gly, Ile, Val

255mg                       Asp, Met

125mg                       Cytosine, Guanosine, Uracil

100mg                       Asn, Leu, His, Lys, Pro, Thr, Try

50mg                          Phe, Thymine, Thymidine

10mg                          CaCl2

2g                               NaAc

10g                             K2HPO4

1g                               Citric Acid

1.3ml                          Trace element solution

36mg                          Ferrous citrate (dissolve in 120ul of conc. HCl)

1ml                             Zn-EDTA solution

1.6g                            Ser

1g                               NH4Cl

Adjust the pH to 7.0

 

Autoclave

 

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.

Grow overnight.

 

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.