10x PBS Recipe for 1 liter

• Dissolve in 800 ml distilled water:
• 80 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44}
• 2 g KCl {Reanal, No.:18050-1-01-38, Kálium-klorid, a.r.; MW: 74.56}
• 14.24 g Na₂HPO₄*2H₂O {Reanal, No.:08973-1-01-38, di-Nátrium-hidrogén-foszfát 2-hidrát, a.r.; MW:177,99}
• 2 g KH₂PO₄ {Reanal, No.:17890-01-38, Kálium-dihidrogén-foszfát, a.r.; MW:136.09}
• Adjust volume to 1 liter with H₂O
• Filtrate through an 0.22um filter and divide in 500ml aliquotes
• Store at room temperature

1x PBS Recipe for 1 liter[137mM NaCl, 2.7mM KCl, 8mM Na₂HPO₄, 1.46mM KH₂PO₄]

• Dissolve in 800 ml distilled water:
• 8 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44}
• 0.2 g KCl {Reanal, No.:18050-1-01-38, Kálium-klorid, a.r.; MW: 74.56}
• 1.424 g Na₂HPO₄*2H₂O {Reanal, No.:08973-1-01-38, di-Nátrium-hidrogén-foszfát 2-hidrát, a.r.; MW:177,99}
• 0.2 g KH₂PO₄ {Reanal, No.:17890-01-38, Kálium-dihidrogén-foszfát, a.r.; MW:136.09}
• Adjust volume to 1 liter with H₂O
• Filtrate trough an 0.22um filter or sterilize by autoclave
• Store at room temperature

PBS-Tween for 1 liter [0.05% Tween 20, PBS]

• Mix on magnetic stirrer:
• 0.5ml Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
• 1 liter PBS

PBS-Tween BSA for 300ml [0.05% Tween 20, 5%BSA, 0.05% azide, PBS]

• Mix on magnetic stirrer:
• 300ml PBS-Tween
• 15g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
• 1.5ml Sodium azide (from 10% NaN₃ stock solution)
• Filtrate through an 0.45um filter
• Aliquot 50ml/falcon
• Store at 4°C

Ca²⁺ Mg²⁺ stock solution for 5ml [0.25M Ca²⁺, 0.07M Mg²⁺]

• Dissolve in 5 ml Milli Q water:
• 0.1838g CaCl₂*2H₂O {Reanal 11024 MSZ 24161-65 Kalcium-klorid, szárított MW: 147.02 CaCl₂*2H₂O}
• 0.0712g MgCl₂*6H₂O {Reanal No.: 20281-1-01-38 Magnézium-klorid 6-hidrát MW: 203.30 MgCl₂*6H₂O}

Ca-Mg-VBS (veronal buffered saline) for 25ml (for serum dilution)[2.5mM Ca²⁺, 0.7mM Mg²⁺, 0.05% Tween 20, 5%BSA, VBS]

• Mix on magnetic stirrer:
  • 5ml 5x VBS
  • 250ul Ca²⁺ Mg²⁺ stock solution [0.25M Ca²⁺, 0.07M Mg²⁺]
  • 1.25g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
  • 12.5ul Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
• Adjust volume to 25ml with Milli Q water
• Filtrate though an 0.45um filter
• Aliquot 1ml/eppendorf
• Store at -20C

Mg-EGTA-VBS solution for 10ml (dilute serum 5x with it)[2.5mM Mg²⁺, 6.2mM EGTA, 5%BSA, 0.05% Tween 20, VBS]

• Mix on magnetic stirrer:
  • 2ml 5x VBS
  • 310ul 0.2M EGTA
  • 25ul 1M MgCl₂ stock solution
  • 0.5g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
  • 50ul 10% Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
• Adjust volume to 10ml with Milli Q water
• Filtrate though an 0.45um filter
• Aliquot 1ml/eppendorf
• Store at -20C

EDTA-VBS solution for 10ml (dilute serum 5x with it)[25mM EDTA, 5%BSA, 0.05% Tween 20, VBS ]

• Mix on magnetic stirrer:
  • 2ml 5x VBS
  • 500ul 0.5M EDTA
  • 0.5g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
  • 50ul 10% Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
• Adjust volume to 10ml with Milli Q water
• Filtrate though an 0.45um filter
• Aliquot 1ml/eppendorf
• Store at -20C

0.2M EGTA stock solution for 10ml

• Mix on magnetic stirrer:
  • 8ml Milli Q water
  • 0.7608g EGTA{ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) Sigma E-4378 LOT 111K5411 FW 380,4}
• Adjust to pH8 with 10N NaOH otherwise it won't disolve (be careful EGTA is an acid)
• Adjust volume to 10ml with Milli Q water
• Store at 4C

1x Veronal buffer (VBS)[145mM NaCl, 1,8mM Na-diethyl-barbiturate (C8H11O3N2Na), 3mM 5,5’-diethyl barbiture acid (C8H12N2O3), pH7.3]

• Dilute 5x Veronal buffer with Milli Q water

5x Veronal buffer for 500ml (5xVBS) [727mM NaCl, 9.12mM Na-diethyl-barbiturate (C8H11O3N2Na), 15.63mM 5,5’-diethyl barbiture acid (C8H12N2O3), pH7.3]

• Make solution 'A':
  • 0.94g Na-diethyl-barbiturate (C8H11O3N2Na){5,5-Diathylbarbitursaure. Na-salz Serva 18797; MW:206.2}
  • 21.25 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44}
  • ~300ml Milli Q water
• Make solution 'B':
  • 1.44g 5,5’- diethyl barbiture acid {5,5-dietil-barbitursav Reanal 04049; MW:184.2}
  • ~100ml Milli Q water
  Boil it otherwise it won’t dissolve, then cool it down
• Mix solution ‘A’ and ‘B’ set pH to 7.3 with app. 50-70μl 10N NaOH
• Fill up until 500ml with Milli Q water, then store it at +4C

Method

• Coat ELISA plate with 50ul/well 0.2-10ug/ml protein (purity should be above 3%) in PBS or carbonate buffer for 2h on R/T or O/N in the fridge
• Wash 3x with PBS-Tween (alternative: following washing incubate in ELISA-blocking buffer for 30min R/T and wash 3x with PBS-Tween)
• Incubate in PBS-Tween diluted antibody/protein solution for 1h at 37C (ideally after 5h reach the equibrium)
• Wash 3x with PBS-Tween
• Develope with TMB
• Add 100ul TMB developing solution to wells
For 1 ELISA plate mix:
• 11ml TMB buffer
• 110ul TMB solution
• 22ul H₂O₂ {Hydrogen peroxide 30% solution Sigma H-1009}
• Add 100ul 2M H₂SO₄ to stop the reaction
• Detect absorbance at 450nm

Carbonate buffer pH 9.5 for 1 liter (for ELISA coat)

• Disolve in 1 liter Milli Q water:
  • 1.6g Na2CO3
  • 2.9g NaHCO3
• Adjust to pH 9.5
• Store at R/T

ELISA blocking-buffer for 10ml [0.05% Tween 20, 5%BSA, 0.05% azide, PBS]

• Mix on magnetic stirrer:
• 10ml PBS-Tween
• 100mg BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
• 50ul Sodium azide (from 10% NaN₃ stock solution)
• Store at +4C

TMB solution for 1ml [10mg/ml TNB in DMSO]

• Disolve in 1ml DMSO {Dimethyl sulfoxide, minimum 99.5% GC Sigma D5879-100ml }
• 10mg TNP {3,3',5,5'-tetramethyl-benzidine Sigma T-2885 FW240.3}
• Store at +4C

TMB-buffer for 1 liter[0.1M Na-acetate, pH 5.5]

• Disolve in 950ml Milli Q water
• 13.6g Na-acetate {Nátrium acetát, kristályvizes CH₃COONa*3H₂O M:136.08 Reanal 14021}
• Adjust pH to 5.5 with app. 700ul cc. Acetic acid {Ecetsav 96% Reanal 0914-1-08-65 M:60.05}
• Adjust volume to 1 liter with Milli Q water

4M H₂SO₄ for 1 liter

• Carefully pour 392ml H₂SO₄ {Sulphuric acid 96% H₂SO₄ Carlo Erba reagents Code no. 410301 (Reanal 17769) FW 98.078} into 608ml Milli Q water (always pour acide into water)

2M H₂SO₄ for 100ml

• mix 50ml Milli Q water + 50ml 4M H₂SO₄ solution

ACK, GKN, RPMI