Oct 28, 2024

Public workspacep-S65-Ub sandwich ELISA

DOI
dx.doi.org/10.17504/protocols.io.n2bvjnq3bgk5/v1
  • Jens Watzlawik1,
  • Fabienne Fiesel1,
  • Wolfdieter Springer1
  • 1Mayo Clinic
Francesca Tonelli
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DOI: dx.doi.org/10.17504/protocols.io.n2bvjnq3bgk5/v1
Protocol Citation: Jens Watzlawik, Fabienne Fiesel, Wolfdieter Springer 2024. p-S65-Ub sandwich ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnq3bgk5/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: July 17, 2024
Last Modified: October 28, 2024
Protocol Integer ID: 104415
Keywords: ASAPCRN, ELISA, PINK1, Ubiquitin, Serine-65
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Abstract
Here we describe an assay to measure the levels of phosphorylation of Ubiquitin at Serine-65 in vivo by enzyme linked immunosorbent assay (ELISA). This assay was previously described by Watzlawik and colleagues [https://doi.org/10.1080/15548627.2020.1834712].
Materials
  • ReagentPhospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAbCell Signaling TechnologyCatalog #62802
  • ReagentUbiquitin Monoclonal Antibody (eBioP4D1 (P4D1)), eBioscience™Thermo Fisher ScientificCatalog #14-6078-37
  • 200 mM sodium carbonate buffer pH 9.7
  • ReagentMULTI-ARRAY 96 Plate Pack, SECTOR PlateMESO SCALE DIAGNOSTICS, LLC.Catalog #L15XA-3
  • ELISA washing buffer
AB
Tris, pH 7.4150 mM
NaCl150 mM
Tween-200.1% [v:v]
• ELISA blocking buffer
AB
Tris, pH 7.4150 mM
NaCl150 mM
Tween-200.1% [v:v]
BSA 1% [w:v]
  • ReagentAnti Mouse Antibody Goat SULFO-TAG LabeledMESO SCALE DIAGNOSTICS, LLC.Catalog #R32AC-1
  • ReagentMSD GOLD Read Buffer AMESO SCALE DIAGNOSTICS, LLC.Catalog #R92TG
  • microplate mixer (USA Scientific, 8182–2019)



Protocols:
Protocols:
3h
3h
Prepare the sodium carbonate buffer, ELISA washing buffer and ELISA blocking buffer.
Use rabbit monoclonal p-S65-Ub antibody as capture antibody and used in a final concentration of Amount1 μg/ml in Concentration200 millimolar (mM) sodium carbonate buffer Ph9.7 and coated DurationOvernight at Temperature4 °C with Amount30 µL per well in a 96-well MSD plate (MSD, MULTI-ARRAY 96-well Plate; L15XA-3).
Wash the next morning MSD plates twice with 0.22-micron filtered ELISA washing buffer using a Amount1200 µL multi-(12)-channel pipettor with Amount300 µL per well. Perform the washing by plate inversion and gentle tapping on paper towels (not by pipette aspiration) and with no incubation time between washes.

• ELISA washing buffer
AB
Tris, pH 7.450 mM
NaCl150 mM
Tween-200.1% [v:v]

Pipetting
Wash
Block the plates with 0.22-micron filtered ELISA blocking buffer for Duration01:00:00 at Temperature22 °C without shaking.

• ELISA blocking buffer
AB
Tris, pH 7.450 mM
NaCl150 mM
Tween-200.1% [v:v]
BSA 1% [w:v]
1h
Temperature
Run all samples in duplicates and diluted in blocking buffer. Load Amount30 µg of total protein per well for all mouse tissues in a total volume of Amount30 µL per well. Adjust the detergent volumes across all samples.
Incubate the antigens for Duration02:00:00 at Temperature22 °C on a microplate mixer (USA Scientific, 8182–2019) at Shaker600 rpm and perform the three washing steps as described before.
2h
Incubation
Wash
Mix
Subsequently add mouse total Ub antibody (clone P4D1; Thermo Fisher #14-6078-37) as detecting antibody in a final concentration of Amount5 μg/ml in blocking buffer in Amount30 µL total volume per well. Incubate detecting antibody for Duration02:00:00 at Temperature22 °C on a microplate mixer at Shaker600 rpm .
2h
Incubation
Mix
Temperature
After three washing steps, add the SULFO-TAG labeled goat anti-mouse antibody (MSD, R32AC-1) in blocking buffer in a final concentration of Amount1 μg/ml using Amount50 µL per well and incubate for Duration01:00:00 at Temperature22 °C on a microplate mixer at Shaker500 rpm .
1h
Incubation
Wash
Mix
After another three washing steps, Amount150 µL MSD GOLD Read Buffer (MSD, R92TG-2) were finally added to each well and the plate being read on a MESO QuickPlex SQ 120 reader.
Perform the data analysis in GraphPad prism 9 or similar software.