Bitan:ELISA: Difference between revisions
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=Ingredients= | =Ingredients= | ||
==Buffers== | ==Buffers== | ||
[[PBS]] | [[PBS]]<br> | ||
[[PBST]] | [[PBST|PBST 0.1%]] [[Tween_20]]<br> | ||
===Coating Buffer=== | ===Coating Buffer=== | ||
Carbonate/bicarbonate (pH 9.6) 100 ml | Carbonate/bicarbonate (pH 9.6) 100 ml<br> | ||
0.293 g NaHCO3 (anhydrous) | 0.293 g NaHCO3 (anhydrous)<br> | ||
0.159 g Na2CO3 (anhydrous) | 0.159 g Na2CO3 (anhydrous)<br> | ||
*Use carbonate/bicarbonate to adjust pH to 9.6*<br> | |||
===Blocking Buffer=== | ===Blocking Buffer=== | ||
2% Amersham ECL Advance Blocking Reagent in PBST | 2% Amersham ECL Advance Blocking Reagent in [[PBST]]<br> | ||
===Phosphate/Citrate Buffer=== | ===Phosphate/Citrate Buffer=== | ||
50 mM citric acid and 100 mM sodium phosphate | 50 mM citric acid and 100 mM sodium phosphate<br> | ||
0.74 g dibasic sodium phosphate (anhydrous) | 0.74 g dibasic sodium phosphate (anhydrous)<br> | ||
1.3 g citric acid (anhydrous) | 1.3 g citric acid (anhydrous)<br> | ||
Dissolve in 50 ml water | Dissolve in 50 ml water<br> | ||
*Adjust pH to 5.0*<br> | |||
*Add 40 μl fresh 30% H2O2 to 100 ml solution before use*<br> | |||
==Reagents== | ==Reagents== | ||
Monoclonal 6E10 | Monoclonal 6E10<br> | ||
=== | Biotinylated 4G8<br> | ||
=== | Streptavidin-HRP<br> | ||
=== | o-Phenylene Diamine (OPD<br> | ||
===1N Sulfuric Acid | 1N Sulfuric Acid<br> | ||
=Recipe= | |||
==Coating== | |||
*Dilute 6E10 (1mg/ml stock) 1:1000 in Coating Buffer to make the Coating Solution<br> | |||
*Add 200ul Coating Solution to each well of a 96-well flat-bottom microtiter plate<br> | |||
*Incubate at room temperature with shaking for 2 hours or overnight in the cold room<br> | |||
==Block== | |||
*Add 200ul Blocking Buffer<br> | |||
*Incubate at room temperature with shaking for at least two hours or overnight in the cold room<br> | |||
==Incubation with Aβ preparation== | |||
*Prepare a 9x two-fold dilution series of the Aβ preparation from 500nM using PBS as diluent<br> | |||
*Add 100ul of each standard to the plate's wells in triplicate or quadruplicate<br> | |||
*Add 100ul of PBS in triplicate or quadruplicate as a control<br> | |||
*Incubate at room temperature for 1.5-2h<br> | |||
==Primary Antibody== | |||
*Dilute Biotinylated-4G8 (1mg/ml stock) 1:1000 in Blocking Buffer<br> | |||
*Add 100ul of the primary antibody solution to each well<br> | |||
*Incubate at room temperature with shaking for 1-1.5h<br> | |||
*Wash the plate with four 10min washes with 110ul PBST per well<br> | |||
==Secondary Antibody== | |||
*Dilute streptavidin-HRP (1.5mg/ml stock) 1:10000 in Blocking Buffer<br> | |||
*Add 100ul of the s-HRP solution to each well<br> | |||
*Incubate at room temperature with shaking for 1-1.5h<br> | |||
*Wash the plate with four 10min washes with 110ul PBST per well<br> | |||
*Tap the plate dry on a pad of Kimwipes<br> | |||
==Color Development and Detection== | |||
*Dissolve four 1mg OPD tablets in 10ml Phosphate/Citrate Buffer<br> | |||
*Add 4ul 30% H<sub>2</sub>O<sub>2</sub><br> | |||
*Add 100ul of the OPD solution to each well<br> | |||
*Incubate for 10min in the dark<br> | |||
*Stop the reaction by adding 100ul 1N Sulfuric Acid per well<br> | |||
*Measure the absorbance at 490nm with the plate reader<br> | |||
Latest revision as of 15:28, 12 November 2009
This ELISA protocol for Aβ concentration works with soluble oligomers and fibrils of Aβ(40) and Aβ(42).
Ingredients
Buffers
Coating Buffer
Carbonate/bicarbonate (pH 9.6) 100 ml
0.293 g NaHCO3 (anhydrous)
0.159 g Na2CO3 (anhydrous)
- Use carbonate/bicarbonate to adjust pH to 9.6*
Blocking Buffer
2% Amersham ECL Advance Blocking Reagent in PBST
Phosphate/Citrate Buffer
50 mM citric acid and 100 mM sodium phosphate
0.74 g dibasic sodium phosphate (anhydrous)
1.3 g citric acid (anhydrous)
Dissolve in 50 ml water
*Adjust pH to 5.0*
*Add 40 μl fresh 30% H2O2 to 100 ml solution before use*
Reagents
Monoclonal 6E10
Biotinylated 4G8
Streptavidin-HRP
o-Phenylene Diamine (OPD
1N Sulfuric Acid
Recipe
Coating
- Dilute 6E10 (1mg/ml stock) 1:1000 in Coating Buffer to make the Coating Solution
- Add 200ul Coating Solution to each well of a 96-well flat-bottom microtiter plate
- Incubate at room temperature with shaking for 2 hours or overnight in the cold room
Block
- Add 200ul Blocking Buffer
- Incubate at room temperature with shaking for at least two hours or overnight in the cold room
Incubation with Aβ preparation
- Prepare a 9x two-fold dilution series of the Aβ preparation from 500nM using PBS as diluent
- Add 100ul of each standard to the plate's wells in triplicate or quadruplicate
- Add 100ul of PBS in triplicate or quadruplicate as a control
- Incubate at room temperature for 1.5-2h
Primary Antibody
- Dilute Biotinylated-4G8 (1mg/ml stock) 1:1000 in Blocking Buffer
- Add 100ul of the primary antibody solution to each well
- Incubate at room temperature with shaking for 1-1.5h
- Wash the plate with four 10min washes with 110ul PBST per well
Secondary Antibody
- Dilute streptavidin-HRP (1.5mg/ml stock) 1:10000 in Blocking Buffer
- Add 100ul of the s-HRP solution to each well
- Incubate at room temperature with shaking for 1-1.5h
- Wash the plate with four 10min washes with 110ul PBST per well
- Tap the plate dry on a pad of Kimwipes
Color Development and Detection
- Dissolve four 1mg OPD tablets in 10ml Phosphate/Citrate Buffer
- Add 4ul 30% H2O2
- Add 100ul of the OPD solution to each well
- Incubate for 10min in the dark
- Stop the reaction by adding 100ul 1N Sulfuric Acid per well
- Measure the absorbance at 490nm with the plate reader
