Patricia L. Clark
last updated: 3/30/00

• Inoculate 100 ml of minimal media with 2 ml of a fresh O/N culture of Salmonella strain 7136.
•Grow at 30°C until 2x108 cells/ml
•Infect with 5-/13- phage to moi=10
•Continue to culture at 30°C for 1.5 hr
•Quickly chill cultures on ice, adding 2 8 ml R buffer ice cubes to each culture to speed chilling; pour into pre-chilled SS34 or GSA bottles; spin @ 5K for 10 min
•Resuspend pellet in 0.5 ml R+OG buffer, split into two microfuge tubes
•Freeze/store in -85°C; or, freeze cells in liq.N2 and store @ -30°C indefinitely


•Make 17 ml sucrose gradients: 16 ml linear gradient of 10-30% sucrose in R buffer, with 0.75 ml 60% sucrose cushion (plus ~0.25 ml sample)
•Thaw frozen cells in RT H2O bath to initiate lysis (can add 75 ul RNase + 75 ul 0.5 M EDTA to destroy ribosomes, if desired; incubate 10 min at RT for complete destruction)
•Spin in microfuge @ 14 K for 1.5 min
•Layer lysate supernatant onto gradients
•Spin 18 K rpm for 18 hr in SW28.1 rotor, or some other combination of speed/time to give w2/sec=2.3x1011 (max speed = 28 K rpm, for 7.25 hr)
•Fractionate gradients using peristaltic pump (pumping from bottom, at 1 ml/min) making thirty (30) 0.57 ml fractions
•Check A260 of fractions: make 12.5-fold dilutions (420 ul R buffer + 80 ul fraction) and measure with Cary UV spec; good A260 for 50S peak ~ 0.6


Day 1
•Coat wells of at least 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 4°C 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 .08 ug/ml 1°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 1°Ab. Also do 1°Ab positive controls (2 per plate), mixed with 250 ul PBS+TWEEN. For all, seal, wrap and store overnight @ 4°C.

*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).

•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 ml PBS+TWEEN, using distriman
•Aliquot 100 ul 2°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


R Buffer: 50 mM TRIS 7.5, 10 mM MgCl2, 150 mM KCl
R+OG: R Buffer plus 80 mM octyl-gluco-pyranoside
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 (use 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 PN