GDP-, GMPCP and GMPPCP-tubulin preparation

Stoichiometrically GDP substituted tubulin preparation

1. Resuspend 20 mg of lyophilized calf brain tubulin in 400 μL of working buffer with 1 mM GDP. Add the buffer in cold a allow sample re-hydration for about 10 minutes (in cold) before pipetting.
2. Load tubulin in a drained centrifuge column of Sephadex G-25 medium (6 x 1 cm), equilibrated in working buffer with 1mM GDP.
3. Spin for 3 minutes at 1300 rpm at 4 ºC in an Eppendorf 5403 centrifuge and recover the flowthrough (remove sugars from the storage buffer)
4. Add GDP up to 10 mM and incubate for 30 min at 4ºC (displace the bound GTP)
5. Load tubulin in Sephadex G-25 column (15 x 0.9 cm) equilibrated in working buffer with 1mM GDP and recover the fractions containing protein (remove the excess of GDP)
6. Centrifuge at 50,000 rpm in a Beckman OptimaTM centrifuge with a TLA-120 fixed-angle rotor (remove protein aggregates)
7. Determine tubulin concentration of the supernatant by UV-VIS absorption spectrometry employing 1cm light path quartz cuvettes. For that prepare 1/200 dilutions in NaPi-SDS buffer and measure the absorbance spectrum (240-330 nm). Calculate tubulin concentration according to Lambert Beer equation (A = c . ε . l), where the molar extinction (ε) coefficient of tubulin at λ = 275 nm is 109000 M-1 x cm-1 (Andreu and Timasheff, 1982).

Stoichiometrically GMPCP and GMPPCP substituted tubulin preparation

1. Resuspend 20 mg of lyophilized calf brain tubulin in 400 μL of working buffer with 2 mM GTP and 1.5 mM MgCl₂ (displace any possible GDP molecule bound to tubulin).
2. Load tubulin in a drained centrifuge column of Sephadex G-25 medium (6 x 1 cm), equilibrated in working buffer with 50 nM of the desired analogue.
3. Spin for 3 minutes at 1300 rpm at 4 ºC in an Eppendorf 5403 centrifuge and recover the flowthrough (remove sugars from the storage buffer).
4. Load tubulin in a Amicon MWCO 50 (Merck-Millipore) concentrator, add 14 mL of working buffer with 50 nM of the desired analogue and, washed tubulin while concentrating the sample (do this 2 times). Final sample volume should be 700-800 μL.
5. Pass the protein through 0.45 μm cellulose acetate microfuge column filters (Costar) (remove aggregates).
6. Add 5 mM of the desired nucleotide analogue and incubated for 30 min at 25 ºC (in the absence of Mg²⁺, these analogues can displace bound GTP).
7. Load tubulin in a Amicon MWCO 50 (Merck-Millipore) concentrator, add 14 mL of working buffer with 50 nM of the desired analogue and, washed tubulin while concentrating the sample (do this 2 times). Final sample volume should be 700-800 μL.
8. Pass the protein through 0.45 μm cellulose acetate microfuge column filters (Costar) (remove aggregates).
9. Centrifuge at 50,000 rpm in a Beckman OptimaTM centrifuge employing a TLA-120 fixed-angle rotor (remove protein aggregates).
10. Determine tubulin concentration of the supernatant as above.

Solutions:

• working buffer (as desired)
• nucleotide stock solutions
• NaPi – SDS Buffer: 10 mM sodium phosphate (1:1 NaH2PO4:Na2HPO4), pH 7.0, 1% SDS

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