Tk:pp-modification

• Borcherding 2003 Affinity membranes as a tool for life science applications, Ann N Y Acad Sci 984:470-9 PMID 12783838
  • 100 mM BP in methanol, 18 hours
  • 8.2 mg/ml GMA + 70 μM BP + 1 mM sodium periodate in 10% methanol
  • 5 minute UV exposure (> 300 nm, 80 mw/cm2)
  • 0.2 mg/ml streptavidin in 100 mM HEPES pH 7.0
  • wash 100 mM HEPES pH 7.0 + 150 mM NaCl + 0.1% Tween-20, 1 hour with agitation
  • final wash with 100 mM HEPES pH 7.0
• US patent 3322661 Yoshikawa67
• US patent 5427779 (example 1, 10) Elsner95
  • The Philips TL20W/09N lamp used here is 20 w UVA (320-400 nm, peak 350 nm). Mehrvar02 measures around 1 mw/cm2 with six.

Blak-ray B-100A lamp, 6 mw/cm2 at 365 nm and 30 cm distance spec sheet

• Test of 5427779 example 10 scheme
  • 10 μg/mL Benzophenone in PBS + 2M CaCl₂ pH 7.3
  • 0.4 μg/mL Psoralen-PEO-biotin
  • UV exposure for 1 hour at (estimated) 1 mw/cm2, equivalent to 10 minutes of the Blak-ray lamp
  • wash 3x with PBS + 2M NaCl + 0.1% NP-40
  • bind streptavidin-HRP conjugate at 0.45 μg/mL + BSA, 1 hour
  • wash 3x as above
  • detect with Ultra-TMB substrate

• Pierce biotinylated HRP reconstitution
  • Pierce 29139 5 mg
  • 1.5 moles biotin/mole HRP
  • Recommended reconstitution at 1 ml DI water, store at -20 in aliquots
  • instead, reconstitute in 50% glycerol + .05% sodium azide to 5 ml (1 mg/ml) store at -20
  • recommended dilutions for antibody detection are 100:1 to 1000:1 of a 1 mg/ml dilution

• Pierce Streptavidin, HRP conjugated (Pierce 21126) 1 mg
  • binds 7 ug biotin/mg streptavidin
  • 2 moles HRP/mole streptavidin
  • Reconstitute in 1 ml of 50% glycerol + .05% sodium azide, store at -20
  • recommended dilution 1-10 ug/ml for staining
  • recommended dilution 0.1 ug/ml for elisa
  • supersignal west pico, dura, femto substrates 1:100,000 to 1:500,000 of a 1 mg/ml solution

• Sigma S4762 Streptavidin 1 mg
  • Reconstitute at 1 mg/ml with 50% glycerol + .05% sodium azide

• Ultra-TMB (Pierce 34028) use
  • Mix neat with detected solution in microplate.
  • Wait 15-30 minutes for color to develop
  • Blue or bluegreen with absorption at 370 and 652 nm. Add equal amounts of 2 M sulfuric acid to stop and convert to a 450 nm yellow absorbing product.

• Pierce EZ-Link Psoralen-PEO3-Biotin (Pierce 29986)
  • Dissolve in DMF or DMSO. Water sensitive. Light Sensitive. Store in light protected tubes.
  • MW 688.79, 36.86A linker arm
  • Pierce recommends dissolving at 20 mM (1.38 mg/100 ul) and mixing at 200 uMolar (1:100 dilution) with 20-100 ng/ul DNA in TE
  • UV exposure (365 nm) for 10 min - 30 min with Philips TL 20W/09 lamp.
  • Ref Zhang Y et al. 2001 NAR 17:143-151

• Ultra-TMB sensitivity test
  • Dilute 1 ul Biotin-HRP into 100 ul TE, then serial dilute 10 ul into 90 ul TE over twelve wells
  • Add 100 ul Ultra-TMB solution
  • E-8 turned dark blue immediately
  • E-9 turned dark blue after 2 minutes
  • E-10 turned light blue after 5 minutes
  • E-11 turned very pale blue after 5 minutes
  • Adding 100 ul 2M sulfuric acid turned the color to yellow, with little density change
  • 1 ul of 1 mg/ml is 1 ug, diluted E-11 is E-16 g
  • MW of biotin-HRP is about 45 KD, so E-16 g is E-16/4.5E4 = 2.2E-21 g
  • This is 2.2E-21 * 6.022E23 molecules = 1.3E3 molecules

• Benzophenone 10 mg/ml in methanol

• PBS + CaCl2 solution precipitated calcium phosphate.
  • The example 10 patent is probably just wrong.
  • Remake solution with 100 mM Tris-HCl, 2M CaCl2