Patricia L. Clark
last updated: 6/6/01
Day 1
* Coat wells of at least four (4) 96-well plates with 100 ul of 1 ug/ml native
tailspike in PBS; leave Column 1 and Rows A and H blank
* Incubate plates @ RT for 3 hr, or store at 4degC for up to two months (wrap
to prevent dehydration)
* Dilute 1-50 ul of fraction* to 250 ul with PBS+TWEEN; mix with 250 ul of 0.08
ug/ml primary Ab (for low-Kd antibodies) in glass fraction tubes. Also do native
tailspike dilution series for standard curve: 0.625 – 0.0391 ug/ml tailspike,
250 ul, each mixed with 250 ul primary Ab. Also do primary Ab positive controls
(2 per plate), mixed with 250 ul PBS+TWEEN. For all, seal, wrap and store overnight
@ 4degC.
* *Deciding how much fraction to use: Because there will be much more tailspike
at the top of the gradient (from native and other released chains), it is necessary
to use far less fraction volume to stay within the reliable region of the tailspike
standard curve. For fractions 1-12, I usually use 1 ul fraction, and then for
fractions 9-30, 15 ul. More fraction may be appropriate for smaller ribosome
preps (i.e., lower A260 values). For conversion of % Recognition to [Native
Tspk Ag], it is really necessary to use % Rec. values below 70% (below 60% is
ideal).
Day 2
* Wash plate wells with PBS+TWEEN, 3x
* Aliquot 100 ul of mixtures into four wells of one column: three coated wells
and one uncoated control (for example, Column 2 Row A-D); repeat for all samples,
standards and blanks. DO NOT put sample in Column 1; save as blank. NOTE: Use
timer to regulate rate of sample addition, starting a new sample every 20 sec.
* Incubate 30 min @ RT; aspirate out samples at same rate as sample addition
(starting new sample every 20 sec); when not aspirating, refill emptied wells
with 200 ul PBS+TWEEN, using distriman
* Aliquot 100 ul 2ary Ab (1:2000 dilution of goat anti-mouse alkaline phosphatase)
into each sample well (do not fill Column 1)
* Incubate 30 min @ RT; wash wells 3x
* Aliquot 100 ul PNPP substrate solution into each well; incubate until bright
yellow (~60 min; check color development with plate reader)
* Read A405 of each plate
REAGENTS
* 10x PBS: 80g NaCl, 2 g KH2PO4, 11.1 g Na2HPO4 (anhydrous), 2 g KCl, to 1 L
w/ H2O; dilute 1/10 to use
* PBS+TWEEN: PBS plus 0.05% Tween-20 (from a 10% stock solution)
* PNPP substrate solution: 1 M ethanolamine, 1 mM MgSO4, to pH 9.8 with HCl;
use 2.5 ml of this to dissolve each 5 mg tablet of PNPP