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|35876S||100 µl (50 tests)||$299.00.0|
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c-Myc (D3N8F) Rabbit mAb (PE Conjugate) #35876
Gallery: c-Myc (D3N8F) Rabbit mAb (PE Conjugate) #35876
Flow Cytometry Triton™ X-100 Permeabilization Protocol – Conjugated Antibody
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS)(#9808): To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde (methanol free).
- Incubation Buffer (1X PBS / 0.5% BSA): Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
- Permeabilization Buffer (1X PBS / 0.3% Triton™ X-100 / 0.5% BSA): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Incubation Buffer.
B. Fixation and Permabilization
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
- Fix for 10 minutes at 37°C.
- Add 5 ml PBS and rinse by centrifugation.
- Resuspend cells in 1 ml Permeabilization Buffer.
- Incubate for 10 minutes at room temperature.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
C. Staining Using Conjugated Primary Antibody
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot 0.5-1x106 cells into each assay tube (by volume).
- If necessary, add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation.
- Add the conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
- Incubate for 30-60 minutes at room temperature.
- Rinse as before in Incubation Buffer by centrifugation.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
posted January 2017