Method Article
Bio-Plex phosphoprotein assay using spinal cords and skeletal muscles of mice
https://doi.org/10.21203/rs.2.12823/v1
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Bio-Plex phosphoprotein assays are magnetic bead-based immunoassays for the detection of intracellular phosphoproteins in cell and tissue lysates. The assay can be readily multiplexed to simultaneously examine the expression levels of numerous phosphorylated proteins.
This protocol was used in our Nature Communications paper.
Bio-Plex ProTM Cell Signaling Assay kit (Bio-Rad Laboratories)
DCTM Protein Assay
Tissue grinder
Scissors
Freezer
Sonicater
Aluminum foil
Eppendorf Tubes Centrifuge
Plate reader for measuring protein concentration
Magnetic washer
Plate shaker
LuminexR 200 xPONETR 3.1 system (Merck Millipore)
Tissue preparation
1. Cut the tissue into small pieces (~3x3 mm) in a 1.5 ml tissue grinder
2. Add 10 times volume of cell lysis buffer and grind the tissue on ice using 20 strokes
3. Freeze samples at -80℃. Freezing and thawing samples helps increase cell lysis effects.
4. Thaw the sample and sonicate on ice (1 min)
5. Centrifuge at 15000xg for 10 min at 4℃
6. Collect the supernatant
7. Measure the protein concentration using DCTM Protein Assay and a plate reader
8. Adjust the concentration of protein to 600µg/ml with cell lysis buffer
9. Freeze samples at -80℃
Assay day1
1. Thaw sample lysates and keep on ice
2. Reconstitute lyophilized cell lysate control with 250 µl of dH2O, vortex for 5 sec to mix and incubate at room temperature for 20 min
3. Centrifuge all samples and lysate controls at 15000xg for 10 min at 4℃
4. Vortex the 20x coupled beads at mid speed for 30 sec and carefully open the cap
5. Dilute coupled beads to 1x with wash buffer, vortex, protect from light with aluminum foil. For the assay in 96wells, mix 288 µl of 20x beads with 5472 µl of wash buffer.
6. Vortex the 1xbeads for 15 sec at medium speed and transfer 50 µl to each well of the assay plate
7. Wash the plate two times with 200 µl wash buffer using magnetic washer
8. Transfer 50 µl of each sample and Bio-Rad cell lysate control to the appropriate wells of the plate. Use detection antibody diluent as the blank.
9. Cover the plate with a new sheet of sealing tape and incubate in the dark for 15-18 hr at room temperature with shaking. Shake at 900-1100 rpm for 30 sec and turn down to 300-450 rpm.
Assay day2
1. Start up, warm up, and calibrate LuminexR 200 xPONETR 3.1 system
2. Bring all buffers and diluents to room temperature
3. Vortex the 20x detection antibodies for 15-20 sec at medium speed
4. Dilute detection antibodies to 1x with detection antibody diluent. For 96wells, mix 150 µl of 20x detection antibodies with 2850 µl of detection antibody diluent.
5. Slowly remove and discard the sealing tape of the assay plate
6. Wash the plate three times with 200 µl wash buffer using magnetic washer
7. Vortex the 1x detection antibodies and transfer 25 µl to each well of the assay plate
8. Cover the plate with a new sheet of sealing tape and incubate in the dark for 30 min at room temperature with shaking. Shake at 900-1100 rpm for 30 sec and turn down to 300-450 rpm.
9. Vortex the 100x Sreptavidin-PE (SA-PE) for 5 sec at medium speed
10. Dilute SA-PE to 1x with detection antibody diluent. For the assay in 96wells, mix 150 µl of 20x detection antibodies with 2850 µl of detection antibody diluent.
11. Slowly remove and discard the sealing tape of the assay plate
12. Wash the plate three times with 200 µl wash buffer using magnetic washer
13. Vortex the 1x SA-PE and transfer 50 µl to each well of the assay plate
14. Cover the plate with a new sheet of sealing tape and incubate in the dark for 10 min at room temperature with shaking. Shake at 900-1100 rpm for 30 sec and turn down to 300-450 rpm.
15. Slowly remove and discard the sealing tape of the assay plate
16. Wash the plate three times with 200 µl wash buffer using magnetic washer
17. To resuspend beads for plate reading, add 125 µl resuspension buffer to each well. Cover the plate with a new sheet of sealing tape and shake at 900-1100 rpm for 30 sec.
18. Slowly remove the sealing tape and place the plate on the reader
The authors have no conflicts of interest to report.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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