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Making Gels and coating with Collagen:
(Preparation of coverslips adapted from Yu-Li Wang et al.)
Cover Slip Glass Activation & Polyacrylamide Preparation
1.
Mark 25mm cover slip opposite the side to be activated
(Either with diamond tip pen or fine point permanent glass pen)
2.
Run cover slip quickly over Bunsen burner flame
3.
Place cover slip down with the flame activated face up
4.
Smear flame activated face with 0.1N NaOH located in the hood, allow to air dry for
10 minutes (use cotton swab applicator)
5.
Once dry smear with 3-aminopropyltrimethoxy silane located in the refrigerator, wear gloves, allow to dry for 5 minutes
* cover entire surface of glass coverslip
6.
Wash with distilled water
7.
Place in 6 well plate with distilled water, then place on shaker for 10 minutes
8.
Aspirate distilled water from the six well plate
9.
Pipette diluted glutaraldehyde onto cover slips under hood let sit for 30 minutes
Dilution Scheme:
From 70% to 0.5% with PBS
14ml PBS
0.1ml glutaraldehyde
(Total volume needed is dependent on how many cover slips are to be coated; ~ 2.5 ml for each cover slip)
 glutaraldehyde is located in the refrigerator in glass vile in 2ml aliquots. Make stock solution in 250 ml of PBS
10.
Aspirated glutaraldehyde off glass coverslip in six well plate
11.
Wash with distilled water while coverslips are still in the six well plate
12.
Remove coverslip from six well plate and allow to air dry
13.
Polyacrylamide preparation (8%-0.06%) for 5ml total solution
Dilution Scheme:
(40% Stock) Acrylamide 5% - 1 ml
(2% Stock) Bis 0.1% - 0.15 ml
Distilled Water - 3.85 ml
14.
Degas polyacrylamide solution for 10 minutes
15.
Prepare ammonium persulfate solution (~1:10 w/v ratio)
Dilution Scheme:
Ammonium persulfate - 10 mg
Distilled water - 100
 l
(stock: 1ml Distilled water and .1g AP and separate in to 100ul aliquots stored in fridge)

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16.
Once coverslips are air-dried take the top cover of the six well plate and place 6 x
10ul drops of water on the bottom of the cover. Place the coverslips on the drops with the activated side facing up
(This is necessary for the coverslips to stay in place when flipping the polyacrylamide solution upside down)
17.
Add fluorescent beads, ammonium persulfate, and TEMED to the degassed polyacrylamide
Dilution Scheme:
Polyacrylamide ~ 5ml
Fluorescent Beads (0.75
 m diameter) – 20
 l
Ammonium Persulfate - 25
 l
TEMED - 10
 l
18.
Place 20
 l of the polyacrylamide solution onto the prepared glass coverslip, then place a 18mm round cover slip over the polyacrylamide and turn top of the six well plate over
19.
Allow solution to polymerize ~ 30 minutes
20.
Place a drop of distilled water or Hepes over the surface of the polyacrylamide glass coverslip
21.
Remove round cover slip from polymerized polyacrylamide
22.
Wash and place on shaker with 50 mM Hepes
23.
In order to activate the polyacrylamide prepare the following
Dilution Scheme:
Sulfo-SANPAH (note must turn off lights when in use) - 0.5mg
50mM Hepes - 1ml
24.
Place 200
 l of the above solution onto polymerized polyacrylamide
25.
Incubate under UV light for 5 minutes
26.
Aspirate off old activation solution and replace with fresh solution and incubate under
UV light for another 5 minutes
27.
Wash each plate with 50 mM Hepes twice and then put on shaker with Hepes for 10 minutes
28.
The collagen can now be placed onto the activated polyacrylamide, and incubated anywhere from 6 hrs to over night at 4°C. (Collagen I: 0.2 mg/ml)
Dilution Scheme:
Collagen stock solution 8mg/ml in Dilute Acetic Acid
Acetic Acid (~.2% v/v in Distilled water)
To make a 50 ml solution of .2mg/ml collagen solution add 1.25 ml of collagen (8mg/ml) to 48.75 ml Acetic Acid (vortex for a few seconds and place in 4-Degree fridge until ready to use.
Notes on SulfoSANPAH: (written by Joyce after discussion with Samuel Zalipsky 10-3-00)
The hydroxysuccinimidyl (NHS) ester can be hydrolyzed either with reaction with water or reaction with a primary amine (e.g. on proteins). Collagen will react with NHS at pH 8 (HEPES buffer). It is best to react overnight in the dark at 4 degC. The low temperature will prevent collagen polymerization.

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If you choose to activate the sulfo-SANPAH before reacting the NHS end, you should prevent the hydrolysis of NHS by (1) using DMSO or acetonitrile (e.g. no water present), (2) lower the pH (~ 5-6), (3) lower the temperature. DMSO is available from Aldrich in 0.5ml, 1ml ampules or in sure-seal bottles that enable you to remove the DMSO by injecting a needle into the seal and using a nitrogen balloon.
It may be possible to store the sulfo-SANPAH in dry DMSO and freeze down in aliquots.
To determine the amount of sulfoSANPAH that has reacted if you first photoactivate, you can use a dye that has an amino group and look at the amount of dye that has depleted from the solution. You may have to dilute the solution because it may saturate the plate reader.
Sigma sells Azocoll (dye-embedded Collagen) and also a FITC-labelling protein kit (under Immunoassays
Section).