- Proteins and Peptides
- Lysates and Cell Lines
Western Blot Protocol
1. Preparation of samples for loading Heat ~50-80ug of sample containing laemmli loading dye (containing SDS) at 90 degrees Celsius for ~2 minutes.
2. Load sample onto a 10% Tris-HCL gel (Bio-Rad pre-cast) and run for ~30 minutes at 200V (or until dye front reaches bottom of gel).
3. Place gel in transfer buffer for 10 minutes (192mM Glycine, 25mM Tris-HCL, 20% Methanol). Pre-soak two pieces of Whatman paper and PVDF, as well. NOTE: The PVDF should be soaked in CH3OH for ~ 1minute, rinsed in ddH20 and then placed in transfer buffer.
4. Transfer the protein from the gel to the membrane using a semi-dry transfer apparatus. Run for 20 minutes at 20V.
5. Block non-specific proteins with blocking buffer #1 (10mM Tris-HCL pH 8.0, 300mM NaCL, 0.025% Tween 20)for 10 minutes. Then continue blocking in blocking buffer #2 (buffer #1 + 15% nonfat dry milk) for an additional hour, gently rocking at room temperature (RT) or overnight at 4 degrees Celcius.
6. Dilute the primary antibody (anti-XRCC3, NB 100-180) in antibody dilution buffer (blocking buffer #1 + 2% milk).
7. Wash the membrane briefly with some blocking buffer #1 and then add your diluted primary antibody.
8. Incubate the primary for 1 hour at room temperature, gently rocking. Again this can be done overnight at 4 Celsius.
9. Wash 3X with blocking buffer #1 for 10 minutes, each, gently rocking.
10. Incubate the diluted secondary antibody (anti-mouse IgG conjugated to HRP), diluted in antibody dilution buffer, for 1 hour at room temperature, gently rocking.
11. Wash 2X with blocking buffer #1 for 10 minutes, each, gently rocking. Wash 1X with blocking buffer #1 for 30 minutes, gently rocking.
12. Develop membrane with your chemiluminescent substrate. NOTE: HEK 293 and HeLa whole cell extracts have been used as positive controls for this antibody.
1. Cells are grown on coverslips in a 6 well dish.
2. Media is aspired off and cells are washed once with PBS.
3. Cells are immersed in 100% methanol for 5 minutes at -20 degrees Celcius.
4. Cells are then blocked in PBS containing 4% BSA overnight at 4 degrees Celcius.
5. Cells are then washed 5 times for 5 minutes each with PBS.
6. Cells are incubated with a 1:500 dilution of purified anti-XRCC3 [NB 100-180] for 1 hour at 37 degrees Celcius in a humid environment using a slide warmer.
7. Cells are then washed 5 times for 5 minutes each with PBS.
8. Cells are incubated with a diluted anti-mouse IgG Alexa 488 conjugated secondary antibody for 1 hour at 37 degrees Celcius in a humid environment using a slide warmer.
9. Cells are then washed 5 times for 5 minutes each with PBS.
10. Coverslips are mounted using Vectashield with DAPI and sealed with polyurethane and stored in the dark at 4 degrees Celcius.
11. Visualization of immunostains was performed by confocal microscopy using a Leica TCS SP2 AOBS instrument.