Jan 31, 2024
Amyloid beta (Aβ) aggregates N-terminal labeling
- Patricia Yuste Checa1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Patricia Yuste Checa, F Ulrich Hartl 2024. Amyloid beta (Aβ) aggregates N-terminal labeling. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3dk4g8j/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: January 25, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94480
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently label protein aggregates at the N-terminal using Amyloid beta as example.
Attachments
974-2531.docx
19KB
Materials
Buffers:
- Labeling buffer: 0.1 Molarity (M) sodium bicarbonate buffer pH 8.3
- 1x PBS pH 7.2
- Dimethyl sulfoxide (DMSO)
Alexa 488 NHS esterThermo Fisher ScientificCatalog #A20000
pHrodo Red SuccinimidylesterThermo Fisher ScientificCatalog #P36600
Amyloid beta (Aβ) aggregates N-terminal labeling
Amyloid beta (Aβ) aggregates N-terminal labeling
30m
Centrifuge Aβ aggregates (e.g., 1 mL at 10 micromolar (µM) ) at 20000 x g, 4°C, 00:10:00 .
10m
Wash the pellet containing the aggregates with 1x PBS 7.2 .
Centrifuge at 20000 x g, 4°C, 00:10:00 .
10m
Wash the pellet with Labeling buffer (refer materials section).
Centrifuge at 20000 x g, 4°C, 00:10:00 .
10m
Resuspend the pellet in 200 µL Labeling buffer.
Dissolve the dye (Alexa488 (A488) NHS ester, Thermo Fisher Scientific, A20000; pHrodo Red Succinimidylester, Thermo Fisher Scientific, P36600) in DMSO.
With a pipette tip gently touch the dye powder which will stick to the tip.
Immerse the tip in some DMSO previously dispensed in a tube.
Repeat the procedure several times until the solution reaches the desired color.
Note
If labeling a protein for uptake assays analyzed by flow cytometry, it is recommended to use A488 because the A488 signal outside the cell can be easily quenched by adding Trypan blue right before measurement. pHrodo red dye is a pH sensitive dye which fluoresces brightly only in acidic environments and therefore can be used to specifically monitor phagocytosis and endocytosis.
Quantify diluted dye concentration by nanodrop. Dilute the sample in water to reach a λ<1 for an
accurate measurement.
Note
Physical characteristics of the dyes to be set in the nanodrop:
Alexa488: Absorbance maximum (λmax): 495 nm; Extinction coefficient (ε): 71,000 cm–1M–1; Correction factor at 280 nm (CF280): 0.11; Correction factor at 260 nm (CF260): 0.3.
pHrodo Red: Absorbancemaximum (λmax): 560 nm; Extinction coefficient (ε): 65,000 cm–1M–1; Correction factor at 280 nm (CF280): 0.12; Correction factor at 260 nm (CF260): 0.36.
Add the corresponding amount of dye to a final protein:dye ratio of 1:10 (e.g., 50 µL of dye at 2.5 millimolar (mM) for 200 µL protein at 50 micromolar (µM) ).
Incubate 01:00:00 at Room temperature in the dark.
1h
Centrifuge at 20000 x g, 4°C for 00:10:00 .
10m
Wash the pellet with 1 mL methanol to remove excess dye (just when labeling with pHrodo dye).
Note
Skip the methanol washing step if using Alexa dye for labeling.
Centrifuge at 20000 x g, 4°C for 00:10:00 .
10m
Wash the pellet 2 to 4 times with 1x PBS 7.2 .
Resuspend the pellet with 1x PBS 7.2 or desired final buffer (e.g., in 200 µL buffer resulting in 50 micromolar (µM) labeled aggregates).
Sonicate labeled aggregates in a Bioruptor sonication bath (Diagenode) (5 cycles of 5 seconds on – 5 seconds off), or similar.
Aliquot, flash-freeze in liquid nitrogen and store at -80 °C .
Note
This protocol can be used for other aggregates like Tau or α-Synuclein aggregates.