Download CASE Kit User Manual

SuperArray CASE™ Kit User Manual
Version 2.2
10/14/2005
16
E. Determine Relative Cell Number:
1. Remove the assay solution as described above.
Wash the wells once with 200 µl of 1X Washing Buffer for 5 minutes with rocking.
Wash the wells once with 200 µl of dH2O for 5 minutes with rocking.
Be sure to remove any excess liquid by tapping plate gently on paper towel.
Air-dry at room temperature for 5 minutes.
2. Add 100 µl of Cell Staining Buffer to each well and incubate for 30 minutes at
room temperature.
WARNING: The Cell Staining Buffer is an intense stain. Avoid contact with skin and
clothing.
3. Rinse the plate twice with 200 µl of dH2O, and then wash the wells twice with 200
µl of dH2O for 5 minutes each with rocking.
NOTE: Wash out as much of the residual color as possible to insure that the OD595
reading will reflect the real cell number.
4. Add 100 µl of 1% SDS to each well and incubate on a rocker for 1 hour at room
temperature.
NOTE: Large air bubbles in the 96-well plate can significantly and artificially inflate your
OD readings. To avoid the appearance of air bubbles, remove a sufficient volume of
every solution from the washing buffer reservoir, avoid pipetting air bubbles, and
examine the plate before placing into your ELISA plate reader. Carefully break any air
bubbles with a pipet tip.
5. Read absorbance at 595 nm on an ELISA Plate Reader.
If the signals are greater than the linear range of the instrument, remove 50 µl of
the 1% SDS solution from every well, and add 50 µl of dH2O to each well. Repeat
the reading of the absorbance at 595 nm on the ELISA Plate Reader.
6. Normalize the antibody reading to the relative cell number:
Divide the OD450 readings for each well by its OD595 reading.
7. To determine the relative extent of target protein phosphorylation, normalize the
phospho-protein specific antibody OD450:OD595 ratio to the pan-protein specific
antibody OD450: OD595 ratio for the same experimental condition.
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