Dec 01, 2023

Public workspaceDirect ELISA

DOI
dx.doi.org/10.17504/protocols.io.261ged83ov47/v1
  • Michael X. Henderson1
  • 1Van Andel Institute
Michael Henderson
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DOI: dx.doi.org/10.17504/protocols.io.261ged83ov47/v1
Protocol Citation: Michael X. Henderson 2023. Direct ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged83ov47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 29, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 91706
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020616
Abstract
This protocol details Direct ELISA.
Attachments
Icon for 911-2362.pdf
911-2362.pdf
169KB
Direct ELISA
Direct ELISA
13h 33m
13h 33m
Dilute Amount100 ng - Amount500 ng of protein of interest (e.g. α-synuclein) in Takeda buffer per well and add Amount30 µL total liquid per well to 384-well Nunc Maxisorp plate.

Pipetting
Seal with removable clear adhesive cover.
Centrifuge the plate at Centrifigation1000 x g for Duration00:01:00 to pull down protein onto the plate. Leave for Duration04:00:00 at Temperature37 °C or DurationOvernight at Temperature4 °C .

4h 1m
Centrifigation
Overnight
Use the plate washer with 5x with Amount100 µL PBST.
Pipetting
Wash
Block with Amount100 µL Blockace per well. Fill wells from the bottom, being sure to avoid leaving any bubbles in the wells.

Pipetting
Seal with a removable clear adhesive cover and leave for Duration04:00:00 at Temperature37 °C orDurationOvernight at Temperature4 °C .

Note
At this point, plates can be stored for up to 1 month at Temperature4 °C if there is a preservative in the buffer.





4h
Overnight
Use the plate washer with 5x with Amount100 µL PBST.

Pipetting
Wash
Use C buffer to dilute reporter antibody. Vortex immediately before pipetting.
Pipetting
Using multichannel, fill 91, dispense Amount30 µL three times.

Pipetting
Seal with removable clear adhesive cover and centrifuge plate at Centrifigation1000 x g for Duration00:01:00 .

1m
Centrifigation
Incubate for Duration04:00:00 at Temperature37 °C or DurationOvernight at Temperature4 °C .

4h
Incubation
Overnight
Use C buffer to dilute HRP-conjugated secondary reporter antibody.
Add Amount30 µL per well. For goat-anti-mouse/rabbit use at 1:5-20K.

Pipetting
Seal with removable clear adhesive cover and centrifuge plate at Centrifigation1000 x g for Duration00:01:00 .

1m
Centrifigation
Incubate for Duration01:00:00 at Temperature37 °C .

1h
Incubation
Use the plate washer with 5x with Amount100 µL PBST.

Pipetting
Wash
Add Amount30 µL TMB reagent per well.

Pipetting
Develop for 10 - 30 min.

Quench using Amount30 µL 10% phosphoric acid per well.

Pipetting
Read plate on the Spectramax or similar plate reader. 384-495 nm for unquenched reactions, 450 nm for quenched reactions.