HPV16 E6 Western Protocol
(Adapted from Arbor Vita)
HNTG buffer:
Stored at 4oC. Make the buffer with the concentrations listed below and add fresh Triton
X-100 and protease inhibitors fresh each time.
50mM HEPES pH7.5
150 mM NaCl
0.1% Triton X-100
1mM EGTA
10% Glycerol
Final HNTG buffer concentrations
50mM HEPES pH7.5
150 mM NaCl
1.1% Triton X-100
1mM EGTA
10% Glycerol
1 mM PMSF
1 x Protease inhibitor
Lysis Conditions for Tissue Culture Cells:
1. Aspirate medium from T.C. plate. Rinse with ice-cold PBS once.
2. Add Xµl (amount varies depending on plate size) of ice-cold HNTG lysis buffer
directly to the cells. Rock the plate at 4°C for 20min.
3. Collect lysate by pipetting and place into an eppendorf. Centrifuge in a
microcentrifuge at maximum speed for 15-20min. (I was told not to scrape the cells, but
I have also tried scraping since the cells do not always lyse well. I don’t think there is too
much of a difference. But scrape lightly and sparingly.)
4. Collect supernatant and store in aliquots at -70°C. Avoid multiple freeze/thaws.
- Add: 200 ml lysis buffer to a 60mm plate
500 ml lysis buffer to a 100 mm plate
1 ml lysis buffer to a 150 mm plate
•
Lysis buffer volume needs to be low vs cell number; lyse for example 20 million cells
in 1 ml lysis buffer! (I think this is flexible)
•
Cell number can be determined via total protein in the lysate: 300 mg protein = 1x106
cells. We find this correlation quite reliable for cervical cancer cell lines.
•
Cervical Cancer cell lines (in our experience) do not like to be overgrown, they will
reduce E6 expression.
Tissue Lysis Conditions:
1. Place 1ml of ice-cold HNTG lysis buffer in a 14ml Falcon tube on ice.
2. Extract tissue and place immediately into the lysis buffer.
3. Homogenize 3X for approximately 10 seconds.
4. Place all contents into an eppendorf. Rotate for approximately 20min. at 4°C.
5. Centrifuge at maximum speed for 15-20min at 4°C.
6. Collect supernatant and store as aliquots at -70°C.
Western Buffers:
• 10X Western transfer buffer without Methanol
390 mM glycine, 480 mM Tris-OH, 0.37% SDS pH 8.3
(for 1 liter, 29g glycine, 58g Tris-OH, 3.7 g SDS)
• 1X Western transfer buffer
1/10 of 10X western transfer buffer, 20% methanol in distilled water.
• Blotting paper
Whatmann 3M, Schleicher and Schull, or equivalent
• Transfer membrane
PVDF (Immobilon-P, Millipore, pore size 0.45µm, cat no. IPVH00010)
• 10X TBS (Tris Buffer Saline)
30g Tris-OH, 87.7g NaCl, 2g KCl; bring up to 1 liter with dH2O, pH 7.4
• 1X TBS
25mM Tris pH 7.4 with 8.77 g/l NaCl and 0.2 g/l KCl (150mM NaCl)
• TBS-T
1X TBS with 0.05 to 0.1% Tween-20
• Blocking buffer
TBS-T with 5% non-fat dry milk and 2% BSA.
• Stripping solution
2% SDS, 100mM βME, 62.5 mM Tris pH 6.8, 60-70oC, 5-30 minutes
Western :
1. Run approximately 100-200µg (tissue culture cells) and 200-250µg (animal tissue) of
protein lysate on a 12% SDS PAGE minigel.
2. Boil samples for 10min.
3. Run gels at 100-105V until dye front passes.
4. Rinse gels with transfer buffer.
5. Transfer at 400mAmps for 1 gel rig for approximately 1.5 hours- 1hr 45min., and
approximately 2hr and 20min for 2 gel rigs.
6. Rinse blot with TBST.
7. Block 4 hours-overnight (overnight is better) at 4°C.
8. Probe with 5µg/mL of E6 antibody in block for 1-2 hours at RT (tissue culture cells)
or overnight (tissue culture cells and animal tissue) at 4°C. I have done 6 hours
incubation for animal tissue and sometimes this is good enough.
9. Rinse with TBST for approximately 30-60minutes, changing the buffer every 510minutes.
10. Apply secondary Goat anti-mouse IgG-HRP (Jackson Immunoresearch, 115-035062, minimal cross-reaction to human, horse and bovine serum proteins) at 1:10,000 in
TBS-T. I did not use this secondary. Instead, I used a heavy-chain specific antibody
from Jackson Immunoresearch, 115-036-071. Incubate 1 hour at room temp rocking.
11. Rinse with TBST for approximately 30-60minutes, changing the buffer every 510minutes.
12. Develop with ECL Plus (Amersham). The band should be above the 15 kd marker.
Sometimes there is a background? band that shows up from time to time.