Flow Cytometry Phosphorylated Protein Fixation/Permeabilization Kit
Cat no : PF00026
Validation Data Gallery
Product Information
Proteintech's Flow Cytometry Phosphorylated Protein Fixation and Permeabilization Kit (Catalog No. PF00026) is for the detection of phosphorylated proteins by flow cytometry. The kit is suitable for experiments where phosphorylated targets compose a majority of the targets to be detected. This kit includes a fixation buffer, a permeabilization buffer, and a staining buffer.
Components
This product contains formaldehyde, methanol, FBS, and sodium azide (≤ 0.09%).
Components | Catalog # | Size | Storage Temperature | Working Temperature |
Fixation Buffer | PF00026-1 | 50 mL | 4℃ | 4℃ |
Permeabilization Buffer | PF00026-2 | 50 mL | 4℃ | -20℃ |
Staining Buffer (10X) | PF00026-3 | 100 mL | 4℃ | RT |
Storage
Stable for 1 year sealed at 4°C. See product label for further details.
Protocol
This protocol was developed for analysis of 1x10^6 cells.
1. Bring all buffers to Working Temperature prior to use (see Kit Components Table).
2. Wash cells with PBS. Discard supernatant.
3. Add 0.5 mL Fixation Buffer. Mix well and incubate at RT for 15 min.
4. Centrifuge at 400-600 g for 5 minutes and aspirate (do not pour off) the supernatant.
5. Add 0.5 mL pre-cooled (-20°C) Permeabilization Buffer. Mix well by pipetting and incubate at RT for 5 minutes.
6. Centrifuge at 400-600 g for 5 minutes and aspirate (do not dump) the supernatant.
7. Dilute the Staining Buffer (10X) to 1X with deionized water.
8. Add 1.5 mL Staining Buffer (1X). Mix well and centrifuge at 400-600 g for 5 minutes and aspirate (do not pour off) the supernatant.
9. Repeat wash step 8.
10. Resuspend cells in 100 μL Staining Buffer (1X).
11. Stain cells with desired antibodies at pre-titrated concentrations.
12. After incubation, wash cells twice as in step 8.
13. If using secondary antibody, dilute the secondary antibody using Staining Buffer (1X). Add 100 μL of diluted secondary antibody and incubate.
14. After incubation, wash cells twice as in step 8.
15. Resuspend cells in 200 μL Staining Buffer (1X).
16. Analyze sample with flow cytometer.
Notes
1. This product contains formaldehyde, methanol, and sodium azide. Take protective measures and avoid contact with eyes and skin.
2. It is recommended to mix with a pipette. Do not vortex and avoid bubbles.
3. During incubation, make sure cells are mixed well and prevent cells from sticking to the side of the tube to prevent false negative populations.
4. Methanol permeabilization of the cell membrane may affect staining of surface markers and may be incompatible with certain fluorophores. It is recommended to predetermine whether any staining of surface markers should be performed before or after the permeabilization step for optimal results.
Cited in Article as
pf00026, Flow Cytometry Phosphorylated Protein Fixation/Permeabilization Kit, Proteintech, IL, USA