Koch Lab:Protocols/Kinesin/Tubulin resuspension and polymerization: Difference between revisions
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==Rhodamine tubulin resuspension== | ==Rhodamine tubulin resuspension== | ||
# Tube usually contains 2λ of a 10 mg / ml solution of tubulin in GPEM + cushion | # Tube usually contains 2λ of a 10 mg / ml solution of tubulin in GPEM + cushion<sup>3, 4, 5</sup> | ||
# Spin down several tubes (say 4 tubes) to get liquid to bottom of tube | # Spin down several tubes (say 4 tubes) to get liquid to bottom of tube | ||
# Combine for total volume of say 8 λ. | # Combine for total volume of say 8 λ. | ||
# Add 8 λ of GPEM | # Add 8 λ of GPEM + cushion [9 parts GPEM, 1 part cushion] | ||
# Store @ -80C in aliquots. I store 1 λ aliquots in 100λ PCR thin-walled tubes. | # Store @ -80C in aliquots. I store 1 λ aliquots in 100λ PCR thin-walled tubes. | ||
<sup>3</sup> Make “GPEM+ Cushion” by mixing 9 parts GPEM with 1 part “Cushion” buffer.<br> | |||
<sup>4</sup> GPEM is made by adding 2 λ of 100 mM Mg-GTP to 198 λ of BRB80<br> | |||
<sup>5</sup> [[User:Steven J. Koch|Steve Koch]] 00:42, 3 May 2009 (EDT): I found the following note in one of my protocols from 2003: "Note: The cytoskeleton storage buffer has the correct amount of GTP, but does not have glycerol. So adding 2λ to 2 λ actually does not produce the “correct” final glycerol concentration. This seems to be OK, though." Whereas above, I say that the storage buffer does have cushion (glycerol). So, I don't know the answer, but this should be easily answerable by contacting cytoskeleton. | |||
Revision as of 21:42, 2 May 2009
Steve Koch 00:32, 3 May 2009 (EDT): I am unsure of the historical details of this protocol. It's the one I used in 2003 and 2004, adapted from George Bachand and probably from Susan Rivera and Andy Boal. They probably followed protocols from Cytoskeleton or the Kinesin Home Page, probably a Howard Lab protocol?
Introduction
This protocol assumes that tubulin has been purchased from Cytoskeleton. Various forms can be purchased. As of 5/2/09, the only two forms we are talking about are lyophilized un-labeled bovine tubulin and rhodamine-labeled bovine tubulin in a 10 mg / ml suspension.
Tubulin dimers are highly unstable, so care should be taken to keep as cold as possible and minimize warm steps except when polymerizing.
Non-labeled tubulin resuspension
- Tube should contain 1 mg of tubulin.
- Dissolve the tubulin in 180λ of cold GPEM buffer1
- Add 20 λ of cold cushion buffer2
- Store @ -80C in convenient aliquots. I do 25λ aliquots in 100λ PCR thin-walled tubes.
1 GPEM is 198λ of BRB80 plus 2λ of 100 mM GTP in 100 mM MgCl2
2 I use cushion buffer from cytoskeleton (BST106), I think it is BRB80 w/ 60% glycerol
Rhodamine tubulin resuspension
- Tube usually contains 2λ of a 10 mg / ml solution of tubulin in GPEM + cushion3, 4, 5
- Spin down several tubes (say 4 tubes) to get liquid to bottom of tube
- Combine for total volume of say 8 λ.
- Add 8 λ of GPEM + cushion [9 parts GPEM, 1 part cushion]
- Store @ -80C in aliquots. I store 1 λ aliquots in 100λ PCR thin-walled tubes.
3 Make “GPEM+ Cushion” by mixing 9 parts GPEM with 1 part “Cushion” buffer.
4 GPEM is made by adding 2 λ of 100 mM Mg-GTP to 198 λ of BRB80
5 Steve Koch 00:42, 3 May 2009 (EDT): I found the following note in one of my protocols from 2003: "Note: The cytoskeleton storage buffer has the correct amount of GTP, but does not have glycerol. So adding 2λ to 2 λ actually does not produce the “correct” final glycerol concentration. This seems to be OK, though." Whereas above, I say that the storage buffer does have cushion (glycerol). So, I don't know the answer, but this should be easily answerable by contacting cytoskeleton.
