96-well phosphoELISA™ Sample Preparation for ... - Invitrogen

BioNote phosphoELISA
96-well phosphoELISA Sample Preparation for Suspension Cells
Products application applies to: Invitrogen’s phosphoELISA kits, designed for measuring the concentration of signaling molecules contained within cell extracts.
Summary: The procedure presented below describes a facile method for studying signal transduction events with suspension cells (Jurkat, Raji,
THP-1, etc.). In this procedure, cells are plated into the wells of 96-well filter bottom plates and stimulated as desired. At the end of the stimulation,
cell culture medium is removed from the bottom of the wells by gentle aspiration using a vacuum manifold. The cells are then washed with PBS,
aspirated, and lysed within the wells by addition of Cell Extraction Buffer. The cell extracts are then assayed using Invitrogen phosphoELISA kits.
Introduction
Invitrogen’s phosphoELISA kits have
been designed to enable the specifi c de-
tection of phosphorylation of key signal-
ing molecules. In the example presented
below, phosphorylation of p38 MAPK
is quantifi ed by phosphoELISA. Threo-
nine 180 and tyrosine 182 are located in
p38 MAPK’s activation loop. Phosphoryla-
tion of these two amino acid residues is
required for full activation of p38 MAPK.
Monitoring the phosphorylation state of
these residues can serve as a substitute
for in vitro p38 MAPK kinase assays, in
which kinase activity is quantifi ed by mea-
suring incorporation of 32 P from γ- 32 P ATP
into protein substrates. Monitoring the
phosphorylation of these two residues
using the phosphoELISA can also serve
as a non-radioactive method for quantify-
ing activation of kinases upstream of p38
MAPK.
Materials
• Sterile 96-well fi lter bottom plates, 1.2 micron
pore size (Millipore Cat. no. MSBVS1210)
• Vacuum manifold (Whatman Cat. no.
7705-0102)
• PBS, ice-cold (Cat. no. 200012-027)
• Cell Extraction Buff er (Cat. no. FNN0011)
• Orbital Plate Shaker (Lab Line Titer Plate
Shaker Cat. no. 4625-EA)
• Invitrogen p38 MAPK [pTpY180/182] phosphoELISA
(Cat. no. KHO0071)
• Invitrogen p38 MAPK Total ELISA (Cat. no.
KHO0061)
Method
1. Enumerate cell density. A hemacytom-
eter is recommended. For most applica-
tions, the cell density should be adjusted
to 0.25-1.0 X 10 6 cells per milliliter cell
culture medium. It is important to note
that this value may require some optimi-
zation for each specifi c application. Cell
doubling time is an important factor to
be considered when adjusting cell density
at the beginning of an experiment.
2. Plate 200 μL of cell culture (i.e., 50,000-
200,000 cells) into the wells of the sterile
96-well fi lter bottom plate. Incubate the
cells for 24 hours at 37°C. The fi lter plate is
designed to retain particles, while permit-
ting the fl ow of liquids from the bottom
of the plate.
3. Stimulate the cells as desired. In the ex-
ample presented below, Jurkat cells were
stimulated with 20 μg/mL anisomycin for
60 minutes at 37°C.
4. At the end of the stimulation, place the
fi lter bottom plate on the vacuum mani-
fold and remove cell culture medium from
the bottom of wells by gentle aspiration.
5. Wash the cells by pipetting 200 μL ice
cold PBS into each well. Remove the PBS
from the bottom of the wells by gentle
aspiration. Repeat two times for a total of
three washings.
Cell Extraction Buff er Formulation
•10 mM Tris, pH 7.4
•100 mM NaCl
•1 mM EDTA
•1 mM EGTA
•1 mM NaF
•20 mM Na 4P2O7 •2 mM Na3VO4 •1% Triton X-100
•10% glycerol
•0.1% SDS
•0.5% deoxycholate
•1 mM PMSF (stock 0.3 M in DMSO)
Protease inhibitor cocktail (Sigma Cat. no.
P-2714), prepared according to the
manufacturer’s guideline as a 10x stock.
Add 100 μL per
1 mL Cell Extraction Buff er.

BioNote phosphoELISA
6. Pipette 300 μL protease-inhibitor-
supplemented Cell Extraction Buff er into
each well. Incubate the plate on ice for 30
minutes.
7. Thoroughly mix the contents of each
well by pipetting up and down 5-6 times.
A multi-channel pipette is desirable for
this application. At this point in the proce-
dure, the extracts are ready for analysis. Al-
ternatively, the extracts may be stored in
the fi lter bottom plate at -20°C for future
analysis. Frozen plates should be thawed
on ice in preparation of completing the
assays.
8. Place the plate on an orbital shaker and
mix for 1 minute.
9. Prepare the 96-well plate for ELISA as
follows:
For ELISA Sample Wells: Pipette 95 μL Stan-
dard Diluent Buff er (included in the ELISA
kits) into the wells of the ELISA plates des-
ignated for samples. Transfer 5 μL cell ex-
tract from the fi lter plate into the sample
wells of the ELISA plates. Place the ELISA
p38 [pTpY180/182] Levels in Anisomycin-Treated
and Non-Treated Jurkat Cells
Cells/ Well
plates on an orbital shaker to thoroughly
mix the contents of the wells.
For ELISA Standard Wells: Prepare stan-
dards as indicated in the assay protocol
and pipette into designated wells.
10. Complete the ELISAs as directed by the
assay protocols.
Results and Discussion
Jurkat cells were seeded into the wells
of the fi lter bottom plate at densities of
50,000, 100,000, and 200,000 cells per 200
μg/mL. The seeded cells were incubated
for 24 hours at 37°C, then treated with
anisomycin (20 μg/mL) for 60 minutes, or
left untreated. At the end of the incuba-
tion period, the cells were lysed by the
method described above, and assayed in
parallel with the p38 MAPK [pTpY180/182]
phophosELISA and the p38 MAPK Total
ELISA.
Figure 1—Anisomycin increases phosphorylation of p38 MAPK at
threonine 180 and 182.
The results presented in Figure 1 were ob-
tained with the p38 MAPK [pTpY180/182]
phosphoELISA (Cat. no. KHO0071). The
data presented show that anisomycin
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terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. F-1028-BN-pELISA US 1006
p38 (pg/mL)
250
200
150
100
50
0
treatment increases the level of phos-
phorylation of p38 MAPK at threonine 180
and 182. The data presented in Figure 1
also show that the quantity of p38 MAPK
[pTpY180/182] measured with this kit is
directly proportional to the number of
cells seeded into the wells of the fi lter bot-
tom plate.
The results presented in Figure 2 were ob-
tained with the p38 MAPK Total ELISA (Cat.
no. KHO0061). This kit was designed to
permit normalization of p38 MAPK phos-
phorylation state. The data presented
show that anisomycin treatment has
no impact on the amount to total p38
MAPK measured in cell lysates, indicating
anisomycin increases p38 MAPK
[pTpY180/182] by enhancing phosphory-
lation state (Figure 1), rather than al-
tering total p38 MAPK level. The data
presented in Figure 2 also show that the
quantity of total p38 MAPK measured with
this kit is directly proportional to the num-
ber of cells seeded into the wells of the
fi l t e r b o t t o m p l a t e .
p38 (Total) Levels in anisomycin-treated
and non-treated Jurkat cells
50,000 100,000 200,000
Cells/Well
Figure 2—Aninomycin treatment does not change the total
p38 MAPK level in cell lysates.
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