Apr 25, 2024

Public workspaceCalf-Intestinal Alkaline Phosphatase Treatment in situ-Killinger 2024

DOI
dx.doi.org/10.17504/protocols.io.5qpvoky7bl4o/v1
  • 1Rush University;
  • 2Rush University Medical Center
Bryan Killinger
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvoky7bl4o/v1
Protocol Citation: Bryan Killinger, Solji Choi 2024. Calf-Intestinal Alkaline Phosphatase Treatment in situ-Killinger 2024. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoky7bl4o/v1
Manuscript citation:
Choi SG, Tittle T, Garcia-Prada D, Kordower JH, Melki R, Killinger BA. Alpha-synuclein aggregates are phosphatase resistant. bioRxiv [Preprint]. 2024 Apr 9:2023.11.20.567854. doi: 10.1101/2023.11.20.567854. PMID: 38645137; PMCID: PMC11030248.
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: April 15, 2024
Last Modified: April 25, 2024
Protocol Integer ID: 98788
Funders Acknowledgement:
NINDS
Grant ID: 1R01NS128467
Michael J. Fox Foundation
Grant ID: ASAP-024442
Michael J. Fox Foundation
Grant ID: MJFF-022480
Abstract
Alpha-synuclein phosphorylated at serine 129 (PSER129) occurs in two pools, non-aggregated (physiological) and aggregated (disease). This protocol allows for the selective dephosphorylation of non-aggregated PSER129 and enhances the specificity and sensitivity immunodetection of aggregated PSER129. Thus, this protocol can be used to differentiate physiological from aggregated PSER129.
Materials
Dilution media:
AB
Nacl 150 mM
Tris-HCl, pH 7.450 mM
Triton-X1000.5%
CIAP buffer:
AB
Nacl100 mM
Tris- Hcl50 mM
Mgcl2, pH 7.910 mM
  • ReagentAlkaline Phosphatase Calf Intestinal (HC), 1,000uPromegaCatalog #M2825

Day 1
Day 1
1d 1h 10m
Wash free-floating tissue (3 x 10 minutes) in dilution media.
  • Dilution media:

AB
Nacl 150 mM
Tris-HCl pH 7.450 mM
Triton-X1000.5%
Wash
Wash free-floating tissue Duration00:10:00 in dilution media (1/3).

10m
Wash
Wash free-floating tissue Duration00:10:00 in dilution media (2/3).

10m
Wash
Wash free-floating tissue Duration00:10:00 in dilution media (3/3).

10m
Wash
Incubate the samples with 1% Triton X-100 in DM for Duration00:10:00 .

10m
Incubation
Wash in DM Duration00:10:00 .

10m
Wash
Wash the tissues in CIAP buffer (2x10 minutes).

  • CIAP buffer:
AB
Nacl100 mM
Tris- Hcl50 mM
Mgcl2, pH 7.910 mM
Autoclave and store TemperatureRoom temperature .
Wash
Wash the tissues in CIAP buffer Duration00:10:00 (1/2).

10m
Wash
Wash the tissues in CIAP buffer Duration00:10:00 (2/2).
10m
Wash
Incubate the tissues with CIAP at a dilution of 1:333 for Duration24:00:00 at Temperature37 °C on a shaker.

  • CIAP concentration per bottle: 20 u/μl (Promega, Cat.# M2825).

  • In Amount500 µL CIAP buffer, add Amount1.5 µL CIAP (30 units).

1d
Incubation
Day 2
Day 2
3h 20m
Wash in DM (2 x 10 minutes).
Wash
Wash in DM Duration00:10:00 (1/2).

10m
Wash
Wash in DM Duration00:10:00 (2/2).
10m
Wash
Heat water bath to Temperature80 °C -Temperature85 °C Duration01:30:00 before the antigen retrieval step.

1h 30m
Place the dish containing sodium citrate buffer in the water bath and heat it for Duration00:10:00 .

  • Sodium Citrate Buffer, pH 6.0 (1L): Amount2.94 g Sodium citrate-Trisodium salt (Dihydrate) in Amount1000 mL DI water. pH 6.0. Add Amount0.5 mL Tween-20. Mix well.

10m
Wash the tissues in sodium citrate buffer Duration00:10:00 .

10m
Wash
Incubate the tissues in the heated sodium citrate buffer for Duration00:30:00 .

30m
Incubation
Cool the dish containing tissues to TemperatureRoom temperature (at least Duration00:20:00 ).

20m
Wash in DM for 10 min x 2 times.
Wash
Wash in DM for Duration00:10:00 (1/2).

10m
Wash
Wash in DM for Duration00:10:00 (2/2) .
10m
Wash
Tissues are now ready for downstream assays.