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