- Proteins and Peptides
- Lysates and Cell Lines
Western blot protocol for detecting the Atg5-Atg12 conjugate
Preparation of Protein Samples
1. Wash cells with PBS once, then add 0.05% trypsin-EDTA for 1 minute at 37 degrees C.
2. Wash the collected cells with cold PBS on ice 3 times.
3. Add Tris-HCl lysis buffer (50 mM Tris-HCl; 150mM NaCl; 1mM EDTA; 100ug/ml PMSF; 1% Triton X-100; 1% protease inhibitor).
4. Rotate for 1 hour at 4 degrees C.
5. Collect the lysate by centrifuging 13,000rpm for 10 min at 4 degrees C.
6. Add Laemmli sample buffer (BIO-RAD, Cat. No. 161-0737) with Mercaptoethanol to the lysates and boil the lysates for 5 minutes at 100 degrees C.
1. Proteins were separated on a 12% SDS-PAGE gel. Approximately 10ug of protein was loaded per lane.
2. Blocking: 5% non-fat dry milk in PBS + 0.1% TWEEN-20 [PBS-T] for 1 hour at room temperature.
3. Incubate the membrane with anti-Atg5 antibody (NB 110-53818) in 5% non-fat dry milk in 1xPBS-T (1:500) at room temperature for 1 hour.
4. Wash the membrane 3 times with PBS-T, 10 min each wash.
5. Incubate the membrane with anti-rabbit secondary antibody in 5% non-fat dry milk in PBS-T at room temperature for 1 hour.
6. Wash the membrane 3 times with PBS-T, 10 minutes for each wash.
7. Add chemiluminescent substrate (PIERCE, Cat. No. 34077) for 5 minutes at room temperature.
8. Develop the film.
1. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes. 2. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
Quench Endogenous Peroxidase:
1 .Place slides in peroxidase quenching solution: 15-30 minutes. To Prepare 200ml of Quenching Solution: Add 3ml of 30% Hydrogen Peroxide to 200ml of Methanol. Use within 4 hours of preparation.
2. Place slides in distilled water: 2 changes for 2 minutes each.
1. Preheat Citrate Buffer. Place 200ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees C.
2. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
3. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
4. Slowly add distilled water to further cool for 5 minutes.
5. Rinse slides with distilled water. 2 changes for 2 minutes each.
1. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap-Pen).
2. Flood slide with wash solution. Do not allow tissue sections to dry for the rest of the procedure.
3. Drain wash solution and apply 4 drops of blocking reagent to each slide and incubate for 15 minutes.
4. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200ul of primary antibody solution to each slide, and incubate for 1 hour.
5. Wash slides with wash solution: 3 changes for 5 minutes each.
6. Drain wash solution, apply 4 drops of secondary antibody to each slide and incubate for 1 hour.
7. Wash slides with wash solution: 3 changes for 5 minutes each.
8. Drain wash solution, apply 4 drops of DAB substrate to each slide and develop for 5-10 minutes. Check development with microscope.
9. Wash slides with wash solution: 3 changes for 5 minutes each.
10. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
11. Wash slides with wash solution: 2-3 changes for 2 minutes each.
12. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
13. Rinse slides in distilled water.
14. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
15. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
16. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
17. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
18. Apply 2-3 drops of non-aqueous mounting media to each slide.
19. Lay slides on a flat surface to dry prior to viewing under microscope.
-Use treated slides (e.g. HistoBond) to ensure adherence of FFPE sections to slide.
-Prior to deparaffinization, heat slides overnight in a 60 degrees C 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.
-200ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections, less than 200ul 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 1-2 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired, increase the time (up to 10 minutes).