Sean Lauber:mOSM ELISA: Difference between revisions
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This protocol requires the special primary antibody in a box in the fridge. This protocol uses reagent diluent from R&D, but you can use sterile filtered 1% BSA (I have tested this and it works fine) | This protocol requires the "special" primary antibody in a box in the fridge. This protocol uses reagent diluent from R&D, but you can use sterile filtered 1% BSA (I have tested this and it works fine) | ||
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1. Dilute primary Ab to a concentration of 8 ug/ml with PBS | 1. Dilute primary Ab to a concentration of 8 ug/ml with PBS | ||
2. Coat an ELISA plate with 100 ul of the diluted | 2. Coat an ELISA plate with 100 ul of the diluted "special" Ab and cover withan acetate plate sealer (Thermo, cat 3501) | ||
3. Let sit at RT overnight | 3. Let sit at RT overnight | ||
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9. Wash 3x | 9. Wash 3x | ||
10. Add 100 ul of biotinylated anti-mOSM (R&D, cat BAF495) diluted to 400 ng/ml in 1x reagent dilutent | 10. Add 100 ul of biotinylated anti-mOSM (R&D, cat BAF495) diluted to 400 ng/ml in 1x reagent dilutent (or 1% BSA) | ||
11. Incubate for 2 hours at RT | 11. Incubate for 2 hours at RT | ||
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12. Wash 3x | 12. Wash 3x | ||
13. Add 100 ul of 1x streptavidin-HRP (R&D, it came with R&D duosets as Part 890803) | 13. Add 100 ul of 1x streptavidin-HRP (R&D, it came with R&D duosets as Part 890803) diluted in 1x reagent diluent (or 1% BSA) | ||
14. Incubate for 20 min at RT in the dark | 14. Incubate for 20 min at RT in the dark | ||
Latest revision as of 06:07, 19 July 2013
This protocol requires the "special" primary antibody in a box in the fridge. This protocol uses reagent diluent from R&D, but you can use sterile filtered 1% BSA (I have tested this and it works fine)
1. Dilute primary Ab to a concentration of 8 ug/ml with PBS
2. Coat an ELISA plate with 100 ul of the diluted "special" Ab and cover withan acetate plate sealer (Thermo, cat 3501)
3. Let sit at RT overnight
4. Wash the plate 3x with 0.05% Tween in PBS (used the ELX50 plate washer (Fisher))
5. Block for 1 hour with 200 ul of 1x Reagent Diluent (R&D, cat DY995) or 1% BSA
6. Wash 3x
7. Add samples and standard (high standard of 2000 pg/ml, 7-point dilution, R&D, cat 495-MO) diluted in 1x reagent diluent (or 1% BSA)
8. Incubate for 2 hours at RT
9. Wash 3x
10. Add 100 ul of biotinylated anti-mOSM (R&D, cat BAF495) diluted to 400 ng/ml in 1x reagent dilutent (or 1% BSA)
11. Incubate for 2 hours at RT
12. Wash 3x
13. Add 100 ul of 1x streptavidin-HRP (R&D, it came with R&D duosets as Part 890803) diluted in 1x reagent diluent (or 1% BSA)
14. Incubate for 20 min at RT in the dark
15. Wash 3x
16. Add 100 ul of freshly prepared substrate solution (H2O2 and Tetramethylbenzidine, R&D cat# DY999 OR BD Bioscience's 555214 which is cheaper and works just as well)
17. After sufficient color development (~15 min at RT in the dark, ie covered with a box) stop the reaction with 50 ul of 2 N H2SO4
18. Read at 450 nm
There can be quite a bit of background with this ELISA and its sensitivity seems to be about 100 pg/ml.
