A sensitive direct human telomerase activity assay Scott B Cohen & Roger R Reddel

Transcription

1 A sensitive direct human telomerase activity assay Scott B Cohen & Roger R Reddel Supplementary figure and text: Supplementary Figure 1 Titration of the sheep polyclonal htert antibody. Supplementary Methods

2 relative activity [antibody] (µg/ml) [antibody] (µg/ml) [antibody] (nm) Supplementary Figure 1. Titration of the sheep polyclonal htert antibody. Antibody was affinity-purified from crude serum over a column of immobilized peptide antigen (ARPAEEATSLEGALSGTRH). Seven 1-ml aliquots of HEK 293 whole-cell lysate ( cell equivalents each) were treated with varying amounts of purified antibody and analyzed with the direct activity assay. For quantitation, the lane with the highest activity is arbitrarily assigned a value of 100.

3 Supplementary Methods MATERIALS REAGENTS Acrylamide/bis-acrylamide solution (40% wt/vol, 19:1 acrylamide/bis, Amresco #0496) Affi-Gel 10 (BioRad # ) Buffer 1: 50 mm Tris-HCl (ph 8.5) Buffer 2: 50 mm Tris-HCl (ph 8.5), 0.5 M NaCl Buffer 3: 20 mm Tris-HCl (ph 8.5), 200 mm KCl, 1 mm EDTA, 0.02% NaN 3 (wt/vol) Buffer TBE: Tris base (90 mm); boric acid (90 mm); EDTA (1 mm) CARPAEEATSLEGALSGTRH ( 90% purity; synthesized by Auspep Pty. Ltd.) Diphtheria toxin (Sigma #D-0564) Fritted glass column with two-way valve (15 mm innner diameter 100 mm length, BioRad # ) Glycine solution (100 mm, ph 2.5 with added hydrochloric acid) NaN 3 solution (2% w/v, 100 ) SPDP (Pierce #21857) Urea (Roche # ) EQUIPMENT Amicon Ultra-4 centrifugal filter (MWCO = 10,000; Amicon #UFC801024) Centricon Plus-20 centrifugal filter (MWCO = 5,000; Amicon #UFC2BCC08) Fritted glass column with two-way valve (15 mm inner diameter 100 mm length, BioRad # ) Large-format DNA sequencing gel (10% wt/vol polyacrylamide; 8 M Urea; 1 Buffer TBE; 400 mm 350 mm 0.2 mm; 32-well comb) PROCEDURE Generation and purification of polyclonal htert antibody 1) Conjugate 20 mg Diphtheria toxin with 10 mg peptide CARPAEEATSLEGALSGTRH ( 90% purity) and SPDP (the C-terminal cysteine reacts with the S-pyridyl disulfide of SPDP). The conjugation of synthetic peptides with immunogenic carrier proteins is a standard procedure with most peptide synthesis companies. 2) Immunize one sheep with the entire Diphtheria toxin-peptide conjugate, administering in four equal doses at weeks 0, 3, 6, and 8. Collect blood serum at week 10. This is typically contracted out through a veterinary service. Our serum was obtained from IMVS Veterinary Services (South Australia). PAUSE POINT Crude serum can be stored at -80 C for at least 5 years. 3) To inactivate proteases, add 0.8 ml 1 M Tris-HCl buffer (ph 8.5) to 40 ml serum and incubate at 55 C for 1 h. Clarify by centrifugation at 3,000g at rt for 15 min. PAUSE POINT The cleared serum can be stored at 4 C overnight; avoid repeated freeze-thaw cycles. 4) Prepare a column of immobilized peptide antigen by combining, on ice and in the following order, 0.5 ml Affi-Gel 10 beads with 2 mg peptide ARPAEEATSLEGALSGTRH ( 90% purity), 0.1 ml CaCl 2 solution (1 M), and 1

4 0.1 ml HEPES-KOH buffer (1 M, ph 7.9). Rotate the suspension at 10 r.p.m. in the cold room (4 C) overnight. CRITICAL STEP 5) Transfer the suspension to a fritted glass column (15 mm inner diameter 100 mm length) and wash the beads by gravity-flow at rt with 20 ml Buffer 2, followed with 20 ml glycine solution (100 mm, ph 2.5), followed with 20 ml Buffer 1. The column can be stored in Buffer TE supplemented with 0.02% NaN 3 at 4 C for at least one year. DO NOT FREEZE. 6) To bind the htert antibody to the antigenic column matrix, add 20 ml cleared serum to the column and rotate at 10 r.p.m. at rt for 2 h. Drain the column, add the remaining 20 ml serum, and repeat binding step. 7) Equip the column with vacuum suction and wash the antibody with ice-cold buffers: 50 ml Buffer % vol/vol Triton X-100, followed with 100 ml Buffer % vol/vol Triton X-100, followed with 50 ml Buffer TE. 8) Elute the antibody in 8 1-ml aliquots of glycine solution (100 mm, ph 2.5), each time allowing the glycine-bead suspension to incubate at rt for 5 min before collecting by gravity-flow into 1.5-ml microcentrifuge tubes containing 0.25 ml Tris-HCl buffer (1 M, ph 8.5). Vortex to neutralize the ph. 9) Pool the 8 fractions into 100 ml Buffer 3. PAUSE POINT The antibody solution may be stored at 4 C overnight. 10) Concentrate the purified antibody to a volume of ~5 ml using a Centricon Plus-20 centrifugal filter (MWCO = 5,000), centrifuging at rt at 3,000g. 11) Reduce the antibody solution further with a Amicon Ultra-4 centrifugal filter (MWCO = 10,000) to a volume of ~ ml. 12) Determine the concentration and yield of htert antibody by UV: 1 OD 280 nm = 0.75 mg antibody/ml. The antibody should be obtained at a concentration of 8-12 μg/μl, providing enough antibody for ~1000 telomerase assays. The htert antibody may be stored at 4 C for at least 6 months without loss of performance. DO NOT FREEZE. DO NOT CENTRIFUGE AT >1000g. Analysis of DNA extension products 13) Pour a large-format DNA sequencing gel (10% wt/vol acrylamide; 8 M Urea; 1 TBE buffer). Refer to supplementary reference 1 for standard protocols regarding sequencing gels. The dimensions of the gels from Figures 2 and 3 are 400 mm length 350 mm width 0.2mm thickness, with a 32-well comb (not a sharktooth comb). 14) Pre-run the gel at 85 Watts for at least 1 h until the gel has reached a temperature of 50 C. 15) Electrophorese 5 μl of each DNA sample at 85 Watts until the bromophenol blue has migrated 80% down the length of the gel. 16) Transfer the gel to filter paper, dry at 80 C for at least 30 min, and then expose the gel to a phosphorimaging screen for a few hours or overnight. 17) Scan the image and quantitate as appropriate. When comparing multiple samples on a single gel, the sample with the highest activity is arbitrarily assigned a value of 100. CRITICAL STEPS STEP 4 For efficient reaction between the activated ester of the Affi-Gel and the NH 2 -terminus of the peptide, precautions must be taken to minimize 2

5 background hydrolysis of the activated ester. The reaction must be maintained cold and must also be performed at high concentrations of reagents. SUPPLEMENTARY REFERENCES 1. Sambrook, J., Fritsch, E.F., & Maniatis, T. Molecular Cloning : A Laboratory Manual (Chapter 6). Cold Spring Harbor Laboratory Press (1989), Cold Spring Harbor, NY. 3