Biology Reference
In-Depth Information
11. Carefully remove each membrane and place into individual
plastic containers with 20 mL of 1× Wash Buffer. Rinse the
4-Well Multi-dish with deionized water and dry thoroughly.
12. Wash each membrane with 1× Wash Buffer for 10 min on a
rocking platform shaker. Repeat two times for a total of three
washes.
13. Dilute the Streptavidin-HRP in Array Buffer 6 using the dilu-
tion factor on the vial label. Pipette 2.0 mL of diluted
Streptavidin-HRP into each well of the 4-Well Multi-dish.
14. Carefully remove each membrane from its wash container.
Allow excess buffer to drain from the membrane. Return the
membrane to the 4-Well Multi-dish containing the diluted
Streptavidin-HRP. Cover the wells with the lid.
15. Incubate for 30 min at room temperature on a rocking plat-
form shaker.
16. Wash each array as described in
steps 11
and
12
.
17. Carefully remove each membrane from its wash container.
Allow excess Wash Buffer to drain from the membrane by blot-
ting the lower edge onto paper towels. Place each membrane
on the bottom sheet of the plastic sheet protector with the
identifi cation number facing up.
18. Pipette 1 mL of the prepared Chemi Reagent Mix evenly onto
each membrane.
19. Carefully cover with the top sheet of the plastic sheet protec-
tor. Gently smooth out any air bubbles and ensure Chemi
Reagent Mix is spread evenly to all corners of each membrane.
Incubate for 1 min.
20. Position paper towels on top and sides of plastic sheet protec-
tor containing the membranes and carefully squeeze out excess
Chemi Reagent Mix.
21. Remove the top plastic sheet protector and carefully lay an
absorbent lab wipe on top of the membranes to blot off any
remaining Chemi Reagent Mix.
22. Leaving the membranes on the bottom plastic sheet protector,
cover the membranes with plastic wrap taking care to gently
smooth out any air bubbles. Wrap the excess plastic wrap
around the back of the sheet protector so that the membranes
and sheet protector are completely wrapped.
23. Place the membranes with the identifi cation numbers facing
up in an autoradiography fi lm cassette.
24. Expose membranes to X-ray fi lm for 15 s to 10 min with mul-
tiple exposure times.
25. Identify the presence of specifi c cytokines and chemokines in
the sample by superimposing the resultant membrane
