RECONSTITUTING LYOPHILIZED TUBULIN
Materials:
- Lyophilized Tubulin (Cat. No. 142001)
- EGTA
- MgCl2
- Liquid Nitrogen
Equipment:
- Microcentrifuge at 4°C
Technical Notes:
- Avoid diluting tubulin beyond its critical concentration
- Work on ice with prechilled buffers and equipment
Protocol:
1. Resuspend
a. Resuspend to 40 mg/ml in ice-cold Milli-Q water
*buffer conditions upon reconstitution with water will consist of 10 mM Sodium Phosphate, 0.5 mM MgCl2 , 0.1 mM GTP, and 0.25 M Trehalose, pH 7.0
*1 mg tubulin = 3 mg powder
b. Add EGTA and MgCl2 to 1 mM each
2. Clarify
a. Clarify at 14,000 rpm for 1 minute at 4°C in a microcentrifuge
b. Collect the supernatant on ice
*tubulin can be diluted, buffer exchanged or cycling as desired at this point
3. Store
a. Flash freeze in liquid nitrogen and store at -80°C
*aliquot so as to avoid repeated freeze-thaw cycles