TERT Antibody (2C4) - BSA Free

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.

Immunofluorescence

1. Cell growth and feeding for IF
A. Seed cells in 4-chamber slides at 20,000 per chamber.
B. Grow to medium confluence
C. Feed with MCDB170+IP at -48 and -24 hr.

2. Fixing cells for IF
A. Wash cells (~70-80% confluent) with 1XPBS
B. Fix slides each in 1:1 ice cold MEOH:acetone and place at -20C for 10 minutes.
C. Store no more than 48 hr in 100% ethanol.

3. IF for hTERT
A. Remove fixative/ethanol from slides.
B. Add 1 ml 2N HCl to each chamber.
C. Incubate for 20 minutes.
D. Remove the HCl and neutralize with 1 ml 0.1 M Na-borate.
E. Incubate for 5 minutes.
F. Remove Na-borate and add 1 ml blocking buffer.
G. Incubate for 2 hr at RT.
H. Prepare NB 100-297 at indicated dilution.
I. Incubate ON at 4C.
J. Wash 4X5 min. in RT PBS.
K. Add secondary (FITC conjugated rabbit anti-mouse IgM).
L. Incubate at RT for 2 hrs.
M. Wash 4X5 min. in 1X PBS.
N. Wash 5 min in 1X PBS with DAPI (1.5 ug/ml).
O. Rinse slides briefly on PBS.
P. Remove chambers from slides.
Q. Mount in Vectashield (Vector catalog # H1200) and observe.

Blocking buffer To 500 ml of 1X PBS:
A. 5 g fish gelatin (Sigma catalog #G7765)
B. 25 ml goat serum
C. 5 g BSA Filter through 0.2 u filter and store at 4C

Immunohistochemistry - FFPE sections

I. Deparaffinization:
A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
B. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.

II. Quench Endogenous Peroxidase:

To Prepare 200 ml of Quenching Solution: Add 3 ml of 30% Hydrogen Peroxide to 200 ml of Methanol.
**Use within 4 hours of preparation

A. Place slides in peroxidase quenching solution: 15-30 minutes.
B. Place slides in distilled water: 2 changes for 2 minutes each.

III. Retrieve Epitopes:
A. Preheat Citrate Buffer. Place 200 ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96C.
B. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
C. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
D. Slowly add distilled water to further cool for 5 minutes.
E. Rinse slides with distilled water. 2 changes for 2 minutes each.

IV. Immunostaining Procedure:
A. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap Pen.
B. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.
C. Drain wash solution and apply 4 drops of Blocking Reagent to each slide and incubate for 15 minutes.
D. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200 ul of primary antibody solution to each slide, and incubate for 1 hour.
E. Wash slides with Wash Solution: 3 changes for 5 minutes each.
F. Drain wash solution, apply 4 drops of Secondary antibody to each slide and incubate for 1 hour.
G. Wash slides with Wash Solution: 3 changes for 5 minutes each.
H. Drain wash solution, apply 4 drops of DAB Substrate to each slide and develop for 5-10 minutes. Check development with microscope.
I. Wash slides with Wash Solution: 3 changes for 5 minutes each
J. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
K. Wash slides with Wash Solution: 2-3 changes for 2 minutes each.
L. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
M. Rinse slides in distilled water.
N. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
O. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
P. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
Q. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
R. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.
S. Lay slides on a flat surface to dry prior to viewing under microscope.

NOTES:
- Use treated slides (e.g. HistoBond) to assure adherence of FFPE sections to slide.
- Prior to deparaffinization, heat slides overnight in a 60 degrees celcius oven.
- All steps in which Xylene is used should be performed in a fume hood.
- For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.
- For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.
- 200 ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts. for small tissue sections less than 200 ul may be used.
- 5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.
- Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-1.5 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired increase time (up to 10 minutes).

Immobilization of Anti-hTERT anibody
All reagents were from the Seize Primary Mammalian IP Kit.
50 ml of mouse ascites (3.3 mg/ml) was diluted with 350 ml of coupling buffer and coupled to 400 ml of AminoLink Plus slurry per the manufactures instructions. Greater than 80% of the protein in the antibody solution were coupled to the beads.


Immunoprecipitation

1. hTERT was synthesized in rabbit reticulocytes using a pET vector and [35S]-methionine was used to allow visualization of the protein.
2. Beads were washed 2X with wash buffer (WB1): 20 mM Tris-acetate, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM MgCl2, 100 mM potassium glutamate, 0.1% IGEPAL, and 1 mM DTT, then blocked twice with 250 mL of blocking buffer (20 mM Tris-acetate, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM MgCl2, 100 mM potassium glutamate, 0.1% IGEPAL, 1 mM DTT, 0.5 mg/mL lysozyme, 0.5 mg/mL BSA, 0.05 mg/mL glycogen, and 0.1 mg/mL yeast RNA) for 15 min at 4C.
3. In between each washing and blocking step the beads were precipitated by centrifugation at 1500g and the supernatant was removed.
4. 50 mL of blocking buffer was then mixed with the 50 mL RNA/protein sample and centrifuged at 17 000g for 10 min at 4C to remove any precipitates.
5. This supernatant was then added to the blocked beads and the samples were mixed on a rotary platform for 2 h at 4C.
6. Following mixing, the beads were washed three times with 325 mL of Wash Buffer #2 (20 mM Tris-acetate, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM MgCl2, 300 mM potassium glutamate, 0.1% IGEPAL, and 1 mM DTT) and twice with 325 mL of TMG (10 mM Tris-acetate, pH 7.5, 1 mM MgCl2, and 10% glycerol).
7. The beads were precipitated by centrifugation at 1500g in between each wash and the supernatant was removed.
8. The beads were then resuspended in 1X SDS gel loading buffer containing 10 mM DTT and analyzed by SDS PAGE.
9. The immunoprecipitation was also performed on 1x10(7) A549 cells.
10. The beads were assayed by TRAP assay.

Results: IP of [35S]-labled hTERT resulted in 10% yield. This is the same efficiency we observed for anti-HA beads used to IP HA tagged hTERT. IP of telomerase from cells allowed isolation of beads that contained telomerase activity.

Conclusion: We successfully immobilized anti-hTERT antibodies on AminoLink beads using the Seize kit from Pierce. These can be used to immunopurify telomerase. The efficiency should be optimized, but the preliminary results are promising.

Protocol courtesy of Pamela K. Dominick and Michael B. Jarstfer from University of North Carolina, Chapel Hill.

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.

**NOTE: This primary antibody is made in mouse and the isotype of the antibody is IgM.