Feb 02, 2024
N_terminal protein 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. N_terminal protein labeling. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxpbrgx1/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: 94602
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently label a protein at the N-terminus using Clusterin protein as example.
Attachments
973-2530.docx
19KB
Materials
Buffers
- Labeling buffer: 0.1 Molarity (M) sodium bicarbonate buffer pH 8.3
- 1x PBS pH 7.2
Alexa 488 NHS esterThermo Fisher ScientificCatalog #A20000
pHrodo Red SuccinimidylesterThermo Fisher ScientificCatalog #P36600
Equipment
Nap-5 column
NAME
Cytiva NAP™
BRAND
45-000-151
SKU
LINK
N-terminal protein labeling
N-terminal protein labeling
1h 30m
Exchange the protein buffer to Labeling buffer using a Nap-5 column (Thermo Fisher Scientific, 45-000-151). Equilibrate the column with 10 column volumes (CV: 1 mL). Load the protein onto the column and elute the protein with the corresponding amount of Labeling buffer following the column manufacturer’s instructions. Collect each eluted drop into an Eppendorf low binding tube. Measure the protein concentration in each fraction by nanodrop and pool the protein-containing fractions.
Note
The protein will be diluted and partially lost in the process. Therefore, it is recommended to start with relatively high protein concentration. Estimated labeled Clusterin yield: around 300 µL at 30 micromolar (µM) labeled Clusterin if starting with 100 µL purified Clusterin at 200 micromolar (µM) .
Dissolve the dye (Alexa488 NHS ester(refer materials section); pHrodo Red Succinimidylester 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 488 nm 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 the diluted dye concentration by nanodrop. Dilute the dye with water for measurement in order to reach an absorbance of 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: Absorbance maximum (λmax): 560nm; 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 diluted dye to the eluted protein to reach a final protein:dye ratio of 1:4-1:10.
Incubate 01:30:00 at Room temperature in the dark.
1h 30m
Remove free dye by using a Nap-5 column, pre-equilibrated with 1x PBS 7.2 buffer or desired final buffer. Collect each eluted drop in an Eppendorf low binding tube. Measure protein concentration in each fraction by nanodrop and pool the fractions containing protein.
Note
Avoid pooling the very last fractions because they may contain free dye.
Measure labeling efficiency (dye molarity/protein molarity) by nanodrop. Quantify independently dye concentration and protein concentration.
Note
If labeling Clusterin, it should be noted that it is a heterodimer containing 2 N-termini and therefore the labeling efficiency may be higher than 1.