10028259

10028259
Bio-Plex Pro Assay Quick Guide
SA-PE Dilution
Volume of
SA-PE, µl
Volume of
1x Assay Buffer, µl
Total Volume, µl
1:10
225
2,025
2,250
Bio-Plex Pro™ RBM Kidney Toxicity Assays
Quick Guide
10. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min at RT.
For Use With
Instruction Manual #
Bio-Plex Pro™ RBM Human, Rat, and Canine Kidney Toxicity Assays 11. Wash the plate three times with 100 µl 1x assay buffer.
10028258
12. After the final wash, resuspend the beads in 100 µl assay buffer. Cover
plate as in Step 4 and shake the plate at 850 ± 50 rpm for 30 sec.
13. Remove the plate seal and read plate at low PMT (Bio-Plex® 200), standard
PMT (Bio-Plex 3D), or default settings (Bio-Plex® MAGPIX™).
This guide can be used to prepare and run a full 1 x 96-well assay plate.
For more information on a given step, refer to the corresponding section
of the complete instruction manual. New users can download the manual,
which includes detailed instructions and a list of kit components, at
www.bio-rad.com/bio-plex.
IMPORTANT! Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and assay variability.
A. Reagent Preparation
1. Reconstitute the following lyophilized reagents in dH20 before use
according to the table below.
Bio-Rad
Laboratories, Inc.
Life Science
Group
10028259 Rev B
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800
Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
Canada 905 364 3435 China 86 21 6169 8500
Czech Republic 420 241 430 532 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0
Greece 30 210 9532 220 Hong Kong 852 2789 3300
Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30
Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapore 65 6415 3188
South Africa 27 861 246 723 Spain 34 91 590 5200
Sweden 08 555 12700 Switzerland 026 674 55 05
Taiwan 886 2 2578 7189 Thailand 800 88 22 88
United Kingdom 020 8328 2000
Sig 1212
Reagent
Volume, µl
Reagent
Volume, ml
Standards mix
150
Blocking buffer
1.5
Control 1
100
Standard diluent
1.0
Control 2
100
Detection antibodies
4.8
a. Allow vial to sit at room temperature for a minimum of 5 min, not to
exceed 30 min.
b. Mix by vortexing at a medium setting.
2. Bring the 10x assay buffer to room temperature (RT).
a. Mix by inversion to ensure all salts are into solution.
b. Prepare 1x assay buffer — dilute 1 part 10x assay buffer with
9 parts of dH20.
Bio-Plex Pro Assay Quick Guide
Bio-Plex Pro Assay Quick Guide
B. Dilution of Standard (1:3 Serial Dilution)
Panel
Sample Dilution
Volume of
Urine Sample, µl
Volume of
Sample Buffer, µl
Human Tox 1
1:4
20
60
2. Transfer the reconstituted standard into the tube labeled “S1.”
Human Tox 2
1:50
10
490
Rat Tox 1
1:2
40
40
3. Add the appropriate amount of the standard diluent into the labeled
tubes according to the table below (this will be sufficient for duplicate
standard curves and blanks).
Rat Tox 2
1:50
10
490
1. Label 8 polypropylene tubes S1 through S8.
Standard
Volume of Standard Diluent, µl
Volume of Standard, µl
S2
100
50 of S1
S3
100
50 of S2
S4
100
50 of S3
S5
100
50 of S4
S6
100
50 of S5
S7
100
50 of S6
S8
100
50 of S7
Blank
100
—
4. Prepare working standards (S2–S8) by serial dilution. Transfer the
appropriate volume of standard into each of the labeled tubes with
standard diluent as outlined above.
5. Vortex each standard at a medium setting before proceeding with the next
serial dilution. Change pipet tip at each dilution step.
Rat Albumin
1:10,000
Canine Tox 1
1:15
Canine Albumin 1:10,000
5 (A. Prepare 1:100) 5 (B. P
repare 1:100)
10
5 (C. Prepare 1:100)
5 (D. Prepare 1:100)
495
495
140
495
495
Note: Controls are ready to use after reconstitution. No dilution is needed.
D. Dispensing of Reagents
1. Add 10 µl of blocker to all wells of the plate.
2. Add 30 µl of the standard, control, sample, or blank to the appropriate well
of the plate.
3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of the
beads to all wells of the plate.
4. Cover plate with plate seal and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.
5. Wash the plate three times with 100 µl 1x assay buffer.
C. Sample Preparation
1. Centrifuge samples at 500 x g for 5 min to remove particulates from all
samples prior to use.
2. Prepare sample dilutions in 0.5 ml or 1.0 ml polypropylene tubes as
required for the assay.
3. Dilution scenarios provided below are sufficient to run each sample
in duplicate.
6. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
7. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do not
aspirate after incubation.
8. Prepare the required dilution of SA-PE as outlined in the following table.
Note: Volumes in the table are for an entire 96-well plate. Smaller volumes
can be prepared, provided that dilution ratios are maintained.
9. Add 20 µl of diluted SA-PE to the required plate wells.
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement