Adenylation of putative RNA ligase proteins in L. tarentolae

Kinetoplast Sample:

To 200 µl of a purified kinetoplast fraction in AMS K-buffer, add 6 µl 10% Triton X-100 (Pierce) to 0.3% final concentration. Homogenize for 1 minute using the Kontes pestle device. Sample is kept on ice Centrifuge in the Microfuge at 4^oC for 1 hr. Remove supernate to a clean tube labeled TS. Can also use a 20S glycerol gradient fraction of TS for cleaner results. Use of TS gives a major 33 kD labeled protein in addition to the p45 and p50 proteins.

Adenylation Reaction:

5 X Buffer
0.1 M Hepes-KOH pH 7.5
0.2 ml 0.5M Hepes PH 7.5
250 mM KCl
83 ml 3M KCl
50 mM Mg2Cl
50 ml 1M Mg2Cl
5 mM EDTA
25 ml 0.2 M EDTA
0.5 mg/ml BSA 25 ml 20 mg/ml

BSA Reactions:

10 ml 5 X buffer
1 ml 0.1 M DTT
1 ml a32PATP
(10 mC/ml)
X ml of Sample
H2O to 50 ml 1 ml

Put at 27°c (water bath) for 30' Add 10-15 ml protein loading dye. Boil for 2 min. Run in a 10% acrylamide - SDS gel at 200-250v until the dye is close to the bottom. Fix gel in 50% methanol - 10% acetic acid for 20-30 min. with gentle mixing. Treat gel with 5% glycerol (with agitation). Transfer to a 3MM filter paper and cover with Saran Wrap. Dry for 1 hr. under vacuum.

Expose to X Ray film or PhosoImager screen overnight. Markers: 15 ml BRL prestained Bench Mark and Rainbow protein MW markers. Acrylamide Stock Solution: 29% acrylamide, 1% bisacrylamide in water. To make 200ml: 58 g acrylamide 2 g bisacrylamide Add water to ~ 120 ml to start dissolving - add another 10-20 ml after a while. Bring vol. to 200 ml using a volumetric flask. Make 10 ml of 10% ammonium persulfate in H2O - 1 g/10 ml. keep at 4°c. To pour the gel (Hoeffer).

15.9 ml HO 13.3 ml 30% acryl. Mix 10.0 ml 1.5 M Tris ph8.8 0.4 ml 10% SDS 0.4ml 10% ammonium persulfate 0.016ml TEMED To make a 1mm gel : Overlay the solution in the gel with water. Remove the water after polymerization- rinse once. Add 5% acrylamide solution.

13.6 ml H2O
3.4 ml 30% acryl. sol.
2.5 ml 1M Tris pH 6.8
0.2 ml 10% SDS
0.2 ml 10% ammonium persulfate
20 ml TEMED

Add 10 well comb - polymerize for 1 hr. Rinse wells just before adding samples. Use Tris-glycine buffer:

5X stock:
15.1 g Tris base
94 g glycine
900 ml H2O.
Dissolve well Then, add 50 ml of 10% SDS. Adjust vol. to 1 liter using a volumetric flask.

1x SDS gel loading buffer

50 mM Tris pH 6.8 1 ml 1M Tris pH 6.8
100 mM DTT. 2 ml 1M DTT
2% SDS 4 ml 10% SDS
0.1% BPB 0.02 g BPB
10% glycerol 2 ml glycerol
11 ml H2O

Freeze in 0.5 ml aliquots