Koch Lab:Protocols/Kinesin/Tubulin resuspension and polymerization
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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?
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.
Tubulin can be purified in-house from various sources, typically bovine brain. Steve's experience is with using commercial tubulin from Cytoskeleton. (Significant hurdles remain to recombinant tubulin production.)
Non-labeled tubulin resuspension
- Tube should contain 1 mg of tubulin.
- Dissolve the tubulin in 180λ1 of cold GPEM buffer2
- Add 20λ of cold Cushion Buffer.
- Store @ -80˚C in convenient aliquots. I do 25λ aliquots in 100λ PCR thin-walled tubes.
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, i.e. 6% (v/v) final glycerin content.
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.
Andy Maloney 01:08, 7 May 2009 (EDT): Some notes about the Cytoskeleton buffers and Koch's above note.
- BST01 is what Cytoskeleton calls PEM but it is just BRB80. I note this because all their buffers for microtubules contain this buffer.
- Koch's note above is talking about Cytoskeleton's BST06 buffer. The one that has GTP but no glycerin. Cytoskeleton calls this GPEM, which is what we are calling it.
- Steve Koch 01:37, 7 May 2009 (EDT): Actually, what I think I meant by this comment is that the tubulin itself was not shipped w/ glycerol. I see now, though, that if we buy the liquid form, we can get it with glycerol. Talking with them on the phone today, though, they said they're moving away from providing the liquid form and trying to sell mostly lyophilized, since it's so much more stable.
- BST05 is Cytoskeleton's GPEM buffer (BRB80 + 1 mM GTP) plus 60% (v/v) glycerin. Koch uses this buffer plus GPEM to dilute the final concentration of glycerin to 6%. Somewhere around 5% glycerin promotes microtubule polymerization, quoted from Cytoskeleton's website.
- Because I store my aliquots in PCR thin-walled tubes, I can pop them right into the thermal cycler w/ hot lid. The hot-lid prevents very small volumes from drying out during polymerization.
- Steve Koch 00:51, 3 May 2009 (EDT): Note: I believe we often did not mix unlabeled with rhodamine-labeled tubulin, and often used 100% rhodamine-labeled tubulin. (Or, rather 100% coming from that cytoskeleton product, probably not 100% efficient, though.) It's probably a much better idea to mix rhodamine-labeled tubulin with un-labeled before polymerizing. At the moment, I don't have the proper ratios for this procedure.
- Pre-heat thermal cycler to 37°C w/ hot-lid ON.
- Heat tubes at 37°C for 20 minutes.
- Add whatever volume of BRB80T (BRB80 buffer with Taxol) appropriate for further experiments. For unlabeled tubulin, I usually add 75 λ of BRB80T to 25 λ of polymerized MTs. For rhodamine tubulin, I think we usually add 100λ of BRB80T. One can use 37°C BRB80T to be extra-careful, but I usually use room temp.
- Procede with experiments, or biospin clean-up
Optional Bio-Spin Column
- The Bio-rad Bio-spin 6 columns can be used to get rid of contaminating Pi (inorganic phosphate) or rhodamine dye, or to shear the MTs
- Steve Koch 00:59, 3 May 2009 (EDT): I never confirmed the ability of the spin column to shear the MTs and I was highly skeptical. Getting rid of Pi is useful for malachite green kinesin activity assays.
- I re-use the bio-spin 6 column, effectively using a column already blocked with tubulin.
- For first preparation of column, I will first block with 200 λ of 2mg / ml (or higher) BSA. Then 500 λ BRB80T, then 100λ of MTs (10 μM), then 4 times 500 λ BRB80T. For an already-used column, I will just do 4 x 500λ BRB80T to equilibrate. Spins are usually in the small fixed-speed table-top centrifuge (Galaxy D?) 30 – 60 seconds.
- Load the MTs (<100λ) onto the column, spin for 60 seconds to recover MTs.
This page was initially prepared based on my Word document notes from Sandia. Ultimately I'd love to have all of them converted to OWW. Until then, I'm manually uploading a few to GoogleDocs. Here's two used to prepare this page: