Protocol: Coupling of glutathione to casein
Version: 1.2
Date: January 2006
Authors: Peter Sehr, Tim Waterboer
Material
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Casein (Sigma C-5890)
PBS
N-Ethylmaleimide (NEM) (Pierce 23030): Stock soln = 20mM in dry DMSO
Sulfo-SMPB (Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate) (Pierce
22317)
o PD10 column (Amersham 17-0851-01)
o Glutathione (Sigma G-6013)
o EDTA
Procedure
1) Dissolve casein in PBS at 37 °C, transfer to 50 ml Falcon tube (150 mg
casein in 25 ml PBS) (= ~0.2 mM cysteine, 2.4 to 3 mM lysine).
Derivatize casein’s cysteine with NEM
2) Add 0.5 ml NEM stock solution (final conc. = 0.4 mM NEM). Incubate 15
min RT, rotating.
Coupling of crosslinker to casein
3) Add 50 mg Sulfo-SMPB (MW = 458.4 final conc. = 4.36 mM). Incubate 30
min at RT, rotating.
4) Remove precipitate by centrifugation (5 min at 6000 rpm), continue with
supernatant.
Removal of free Sulfo-SMPB
5) Load 2.5 ml supernatant on PD10 column (equilibrated with PBS), discard
flow-through, add either 0.5 + 2.5 ml or 3 ml PBS, collect eluate.
Concentration of product increases if first 0.5 ml of eluate is discarded, but
work-load also increases.
6) Wash column with 15 ml PBS. Repeat procedure for next 2.5 ml
supernatant.
Binding of glutathione to casein-MPB
7) (25) 30 ml reaction batch + (5) 6 ml glutathione solution (120 mM in PBS +
10 mM EDTA, adjust to pH 7.4 with NaOH finally 20 mM), 60 min RT,
rotating.
8) Remove free glutathione with PD10 column (see above).
Store in aliquots at -20 °C, avoid repeated freezing/thawing.
Casein: assumption 1 cysteine, 12 to 15 lysine residues per molecule
Protocol: Coupling of glutathione-casein to carboxylated
(COOH) Luminex beads
Version: 1.2
Date: January 2008
Author: Tim Waterboer & Joseph Carter
Material
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COOH beads
Amicon 1.5 ml microfilters 0.45 tubes
Activation buffer (AP): 0.1 M Sodium phosphate pH 6.2 ± 0.2
Coupling buffer (KP): 50 mM MES pH 5.0 (adjust with NaOH)
Wash buffer 1 (WP1): PBS, 0.05 % Tween 20, 50 mM Tris pH 7.4 ± 0.1
Wash buffer 2 (WP2): PBS, 0.05 % Tween 20 pH 7.4 ± 0.1
Blocking buffer (BP): PBS, 1 mg/ml Casein pH 7.4 ± 0.1
Storage buffer (LP): PBS, 1 mg/ml Casein, 0.05 % Sodium azide pH 7.4 ± 0.1
NHS (N-Hydroxysuccinimide)
EDC (1-(3-Dimethylaminopropyl)-3-Ethylcarbodiimide)
Water-free DMSO
Glutathione-Casein
96 well plates
Devices
• Tabletop centrifuge
• Vortexes
• Shaker
• BioPlex 200
Preparation and comments
• Luminex beads are light sensitive. Therefore, keep them in the dark whenever
possible!
• Be careful when resuspending beads in small volumes. Don’t vortex too long;
otherwise the beads will be rather in the lid of the tube than in its bottom.
• The beads have a density of 1.05 mg/ml, i.e. they are not much heavier than
water. After centrifugation steps, quickly remove the supernatant, otherwise the
pellet may loosen from the tube wall (depends on buffer).
• The beads mustn’t dry! When removing supernatants, always leave some 50 µl
on the beads if not indicated otherwise.
• EDC is moisture sensitive. Avoid air contact. Store aliquots at -20 °C and use
them only once. Same applies to NHS aliquots.
• Allow all reagents to reach RT before use.
Activation of beads
1) Vortex bead stock.
2) Transfer 0.3 ml of the homogeneous bead solution into an Amicon 1.5 ml
microfilters 0.45 tube, centrifuge 2 min at top speed (all centrifugation steps
are at the highest possible centrifuge speed), and discard supernatant.
3) Add 500 µl AP. Centrifuge 2 min, discard supernatant.
4) Add 200 µl AP to the filtration tube. Work quickly!
5) Immediately prior to use, dissolve an EDC aliquot in AP (50 mg/ml), vortex.
6) Immediately prior to use, dissolve an NHS aliquot in water-free DMSO (50
mg/ml), vortex.
7) Add 25 µl of the NHS solution to the beads, vortex.
8) Add 25 µl of the EDC solution to the beads, vortex.
9) Incubate beads under shaking (200 rpm) for 20 min in the dark at RT.
Protein coupling
10) Adjust Glutathione-Casein to 250 µg/ml in KP.
11) Centrifuge beads for 2 min, discard supernatant.
12) Add 500 µl coupling buffer (KP) and centrifuge for 2 min, discard
supernatant.
13) Add 250 µl of the Glutathione-Casein solution. Coupling takes place under
shaking (200 rpm) for 2 h in the dark at RT.
14) Centrifuge beads for 2 min, discard supernatant.
15) Add 500 µl WP1. Incubate for 15 min shaking (200 rpm) in the dark at RT.
16) Centrifuge beads for 2 min, discard supernatant.
17) Add 500 µl WP2. Centrifuge for 2 min, discard supernatant. Repeat washing
step and thoroughly remove washing buffer.
18) Make the volume of bead equal to the volume initially removed from the
tube with LP.
19) Store beads at 4 °C in the dark.
Count beads using hemocytometer
Each 16 square quadrant and the 25 square center are 1.0 X 10 -4 ml. Dilute the
beads 1:10 in water. Count between 1 and 5 quadrant so that at least 100 beads
are counted. Divide the number of beads by the number of quadrants and by 1.0
X 10-4 ml
Counting beads using the Luminex
Dilute 1 µl resuspended bead suspension in 96 well plate with 100 µl BP. For the
measurement in the Luminex analyzer (see corresponding protocol). Program
the machine read a bead set that is not included so that it will read the full 200
sec. The approximate number of beads is 3.5 times the number read.
Anti-tag assay
To control for successful bead coupling, load 1 µl beads (or 10,000 beads to
additionally control for correct counting) for 1 h under shaking in the dark with 1
ml of bacterial lysate diluted to 1 mg/ml in BP. The lysate must contain over
expressed protein with C-terminal SV40 tag epitope. After loading, wash beads
three times by pelleting, removing the supernatant, and adding 1 ml BP. After
third wash, resuspend beads in 165 µl BP. Apply each 50 µl (= 3,000 beads) to
each one well of a 96 (half) well plate, add 50 µl of anti-tag mAb (1:1000),
incubate plate for 30 min shaking in the dark, add 50 µl of Strep-PE (1:1000) and
incubate for 20 min shaking in the dark.
Read on BioPlex 200 – Can also recheck the number of beads by including
another bead type in the program that is not used in the experiment.
Protocol: Handling of BioPlex 200 analyzer
Version: 2.1
Date: January 2008
Author: Joseph Carter
Material
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Sheath Fluid, Isopropanol, Millipore water
Bleach solution (10 %)
Calibration Beads
Filter Plates
BioPlex Plate
Devices
• Vortexer
• BioPlex 200 Analyzer, XY Platform, Sheath Delivery System
Startup procedure
1) Switch on BioPlex 200, XY Platform and Sheath Delivery System
2) Switch on PC and monitor. Log in and open the BioPlex software
3) Put Water and isopropanol in the labeled well of the BioPlex plate and put
the plate into the XY platform
4) Run “Startup”. This will also start the Laser warmup procedure which takes
about 30 min
5) Run “Warmup”. This program also has to be run if the machine has been
idle for over 3 hrs
6) Vortex the calibration beads and add 5 drops of each to appropriate wells of
the BioPlex plate
7) Run “Calibrate”
8) Open a “Program” and select bead sets and wells
9) Insert plate
10) Run the program
11) Save the data file and run “Wash” procedure. If an entire plate has been
read then run the “Wash” procedure twice
12) Either run a new or modified program or run the “Shut Down” program. Prior
to running “Shut Down” add 10% bleach to BioPlex plate
Protocol: Luminex Serology Assay
Version: 1.2
Date: January 2008
Author: Tim Waterboer & Joseph Carter
Material
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GC-beads
Bacterial lysates, sera
1.5 ml Eppendorf tubes (e.g., Starlab)
Blocking buffer (BP): PBS, 1 mg/ml Casein pH 7.4 ± 0.1
Storage buffer (LP): BP, 0.05 % sodium azide
Detection system (e.g. goat-α-human IgG-Biotin & Streptavidin-PE)
96 well wash plates (Millipore)
96 well polypropylene plates
Casein, Polyvinylalcohol, Polyvinylpyrrolidone, CBS-K
Devices
• Luminex 100, XY Platform, Sheath Delivery System
• vortexer
• Table centrifuge
• Ultrasonic bath
• Shaker
• Vacuum manifold
Loading of GC-beads with antigen
1) Dilute the bacterial lysates to 1 mg/ml in BP in Starlab Eppendorf tu rotating
bes. 1 ml of the dilution is sufficient for up to 3 million beads.
2) Centrifuge the GC-beads for 2 min at 13,000 rpm and thoroughly vortex
them.
3) Pipet the necessary amount of beads (see exemplary calculation) directly in
the lysate dilution (one bead sort per antigen). Incubate on a shaker (200
rpm) for 1 h at RT in the dark.
4) Centrifuge the beads for 2 min at 13,000 rpm, discard the supernatant, add
1 ml of BP and vortex until the beads are resuspended. Repeat washing
procedure twice (three times total).
5) After the third wash, remove the supernatant thoroughly and add 200 µl of
BP
Preincubation of sera
6) Dilute sera (1:50) in blocking reagent (BP containing 2 mg/ml GST tag
lysate, 0.5 % Polyvinylalcohol, 0.8 % Polyvinylpyrrolidone, and 2.5 % CBSK) (use 2 l serum into 98 l block) in a 96 well polypropylene plate
7) Incubate the serum dilution on a shaker (200 rpm) for 1 h at RT in the dark
Equilibration of wash plates
8) Incubate the wash plates with 100 µl BP per well for 10 min at RT.
Afterwards, remove the buffer with the vacuum manifold and dry the plate
using the hammer (2-3 times).
Incubation of beads and sera
9) Vortex beads for 30 sec and sonicate for 1 min
10) Repeat above 3-6 times until bead suspension is completely homogeneous
11) Combine bead suspensions in an appropriate vessel. In order not to loose
the remaining beads, add adequate volume (e.g. 500 µl) of BP to the
Eppendorf tubes, vortex, and transfer the buffer to the vessel with the
multiplex mix.
12) Add BP to the needed volume (see exemplary calculation). Vortex the bead
mix and fill each well of the equilibrated wash plates with 50 µl bead mix.
Transfer each 50 µl of the serum dilutions from the polypropylene to the
wash plates. Incubate the plate on a shaker (200 rpm) for 1 h at RT in the
dark or incubate at 4o overnight followed by 30 min on a shaker at RT
Incubation with secondary antibody
13) Remove serum from the wash plates using the vacuum manifold. Wash the
plates three times with 100 µl BP per well. Dry plate using a hammer.
14) Add secondary antibody (e.g. goat-α-human IgG-Biotin 1:1000 in BP) and
incubate the plate on a shaker (200 rpm) for 30 min at RT in the dark.
Incubation with Streptavidin-R-Phycoerythrin (Strep-PE)
15) Remove the secondary antibody using the vacuum manifold.
16) Wash the plates three times with 100 µl BP per well. Dry the plate using the
hammer.
17) Add Strep-PE (light sensitive!) diluted 1:1000 in BP and incubate the plate
on a shaker (200 rpm) for 30 min at RT in the dark.
18) Wash plate three times and dry. Add 100 µl of BP per well (LP if plates are
stored o/n at 4 °C and read the next day).
19) Shake for 5 min at RT in the dark to resuspend the beads and perform
measurement in the BioPlex analyzer.
EXEMPLARY CALCULATION
The reactivity of 120 sera against 5 different antigens plus GST-Tag is to be
determined. Per data point 2,750 beads are needed. Therefore, 330,000 beads
from 6 different bead sorts are needed.
Loading of GC-beads with antigen
The concentration of beads after the coupling reaction with GC has been
determined. A typical value is 15,000 beads per µl. For 330,000 beads (330,000 /
15,000 =) 22 µl of bead suspension have to be loaded with antigen.
Incubation of beads and sera
After washing the antigen loaded beads 200 µl of buffer have been added. For
120 sera (120 x 50 =) 6,000 µl bead mix are needed. The empty Eppendorf tubes
are washed with each 500 µl BP, the final mix looks therefore like this:
6 x 200 µl bead suspension = 1.2 ml
6 x 500 µl BP wash volume = 3.0 ml
Add BP 1.8 ml
6.0 ml