If you are going to be doing many assays, I recommend doing a 500 μL or 1000 μL translation. This does get expensive since a rabbit kit costs about $400. The kit can make a total of 2000 μL of translation. The translation kit is purchased from Promega. The kit is called the TNT Quick Coupled Transcription/Translation system catalog # L1170. The human TERT gene is in the pCite vector and was given to me by Carol Greider. The gene encodes a 2X HA tag on the C-terminus of the hTERT gene. After the translation, I purify the telomerase using HA tag antibody Agarose from Santa Cruz Biotechnology. HA-probe (F-7) catalog# sc-7392 AC. With this catalog #, you get 1 ml HA Tag Antibody Agarose ( 1 ml of a 50% v/v suspension)
A typical translation:
|TNT Quick Master Mix (stored on bottom shelf Revco room 344)|
|8 μL||35S methionine (perkin Elmer) (stored on Top shelf Revco room 344)|
|10 μL||1 mM methionine (supplied with kit)|
|10 μL||PCR enhancer (supplied with kit)|
|10 μL||1.0 μg/μL hTERT plasmid (in H2O)|
|10 μL||1.0 μg/μL human telomerase RNA (in H2O)|
|500 μL||total volume|
Incubate 90 mins to 2 hrs at 30°C.
If you are using 35S in your translation, anytime during the translation, remove 2 μl to count. I do this two times. The numbers you get can be used later to determine the amount of human TERT made during this translation.
Immunopurify the human telomerase with HA beads washed with 1X human telomerase buffer ( without glycerol or KCl). For a 500 μL translation, I add 100 μl of 1:1 HA bead slurry (see below for HA bead prep). This should give you enough telomerase to do about 15 telomerase reactions. Depending on how quickly you want to expose your phosphor-screen, you will be able to dilute the beads even more.
After adding the HA beads to the translation, nutate, rock or rotate the tube at 4°C for a minimum of 2 hr. I have gotten similar recovery of telomerase if a do this step for 2 hours or overnight.
Pellet the beads at 3500 rpm for 1 min in a table-top centrifuge (Eppendorf centrifuge model 5424 or 5415D.
After centrifugation, let the tube stand on ice for an additional minute or two to let the beads settle from the side of the tube.
Pull off the liquid, trying not to pull up the settled beads. If it is difficult to see the interface between the beads and the blood, try to angle the tube a little. Sometimes this helps you identify the interface. After the first wash, this becomes less difficult.
Add 1 ml of 1X human telomerase buffer (with 30% glycerol) to the beads. Agitate the tube to resuspend the beads.
Centrifuge in table top centrifuge at 3500 rpm. Let beads settle as before. Remove the liquid from the beads.
Do this three or four times ( until you get the red out) with 1X human telomerase buffer (with 30% glycerol).
After the final wash, make a 1:1 slurry of beads with 1X human telomerase buffer (with 30% glycerol). If you used 35S in your translation, count 2 μl of the slurry to get yield of telomerase. I do two 2 μl aliquots.
Use beads immediately, or quick-freeze in Liquid N2 and store at -80°C
Beads are sticky. I cut the ends of my p-20 and p-200 tips to increase the orifice of the tip. I usually cut off 2-3 mm of the tip with a razor blade. If you want to use p-10 tips, cut the tip off at the first step in the tip. This should give you a large enough hole in the tip for the beads.
HA bead Wash procedure:
HA beads are washed by first centrifuging the bead slurry at 3500 rpm in a 1.6 ml Eppendorf tube for 1 min in a table-top centrifuge (Eppendorf centrifuge model 5424 or 5415D ( I usually wash a 1ml aliquot)).
After centrifugation, I let the tube stand on ice for an additional minute or two to let the beads settle from the side of the tube.
Pull off the liquid, trying not to pull up the settled beads.
Add 1 ml of 1X human telomerase buffer( without glycerol or KCl) to the beads. Agitate the tube to resuspend the beads.
Centrifuge in table top centrifuge at 3500 rpm. Let beads settle as before, before removing the liquid from the beads.
Do this three or four times with 1X human telomerase buffer (without glycerol or KCl)
After the final wash, make a 1:1 slurry of beads with 1X human telomerase buffer (without glycerol or KCl). If you do not use all the beads in your immunopurification, store them at 4°C.
human telomerase assays:
Buffers and solutions
10X human telomerase buffer:
To make 20 mLs of 10X human telomerase buffer
|1 M Tris-HCL pH8.0||
|3 M KCl||3.34|
|1 M MgCl2||0.2|
|1 M Spermidine||0.2|
|14.3 M β-mercaptoethanol||0.070|
1X human telomerase buffer: (5 mls 10X human telomerase buffer and 45 mls H2O.)
1X human telomerase buffer with 30% glycerol (5 mls 10X human telomerase buffer, 15 g Glycerol, H2O to 50 mls.) I weigh the glycerol on a table top balance. I find this easier than pipeting.
20X dNTP’s (made by adding 5.8 μL 1 mM dGTP, 10 μL 100 mM dTTP, 10 μL 100 mM dATP into 74.2 μL H2O). Stock dNTPs are from GE Healthcare catalog# 28406551.
0.33 μM 32PdGTP (2 μL of 3,000 Ci/mMole 32P dGTP from Perkin Elmer added directly to assay) Stored in refrigerator in room 344
Total [dGTP] = 3.23 μM (0.33 μM 32PdGTP + 2.9 μM cold dGTP)
Stop buffer (made by adding 10 μl of 20 mg/ml glycogen to 1 ml 3.6 M NH4AC). You will need 100 μl of stop buffer for each telomerase reaction.
Sample prep buffer (0.1X TBE, 50 mM EDTA, 0.1% Bromophenol blue, 0.1% Xylene Cyanol, 93% formamide)
|Telomerase bead slurry||6 µl|
|10X human telomerase buffer||1.4 µl|
|32P-dGTP (3000 Ci/mMole)||2 µl|
|20X human dNTPs||1 µl|
|20 µM primer a (TTAGGG)3||1 µl (primer concentration can vary)|
|Total volume||20 µl|
Incubate 1hr at 30°C
Add 100 μl of stop buffer (3.6 M NH4AC, 20 μg glycogen).
Add 500 μl ethanol
Incubate -80°C for a minimum of 45 minutes (This is a good place to stop, and let the precipitation go overnight)
Pellet 13k RPM for 15 minutes at 4°C.
Pour off liquid, add 1 ml 70% ethanol. Invert tubes
Pellet 13k RPM for 10 minutes at 4°C.
Pour off liquid, dry pellets in speedvac.
To each tube, add 10 μl H2O, followed by 10 μl of sample prep buffer. Flick tubes to mix.
Incubate tubes 5 minutes 95°C.
Pellet insoluble material by centrifugation at 13K RPM 5 minutes at room temperature.
Pipet off 10 μl of sample to load on 10% acrylamide/ 7M urea gel.
Before loading the sequencing gel, the gel is pre-run at 90W for 45 minutes. After loading samples, the gel is run at 90W for about 1.5-2 hours. I usually run the Bromophenol blue dye to the bottom of the gel.
For additional information, contact Art Zaug.