May 23, 2023
Cell lysis and immunoblotting for protein and phospho-protein quantification
- Dan Dou1,2,
- C. Alexander Boecker3,
- Erika L.F. Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 3Department of Neurology, University Medical Center Goettingen, 37077 Goettingen, Germany
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur 2023. Cell lysis and immunoblotting for protein and phospho-protein quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8j5zrg2w/v1
Manuscript citation:
Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur EL, Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell reports 42(5). doi: 10.1016/j.celrep.2023.112448
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: October 13, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71304
Keywords: Lysate, Immunoblot, Western, Phospho, LRRK2, ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Here, we describe the procedure by which human iPSC-derived neurons or mouse embryonic fibroblasts (MEFs) were lysed and probed for levels of proteins of interest using Western blot.
Attachments
552-1148.pdf
77KB
Materials
Reagents
- RIPA buffer
| A | B | |
| Tris-HCl | 50 mM | |
| NaCl | 150 mM | |
| Triton X-100 | 0.1% | |
| Deoxycholate | 0.5% | |
| SDS | 0.1% |
- HALT phosphatase and protease inhibitor cocktail (100x)Thermo Fisher ScientificCatalog #78442
- Microcystin-LR Microcystis aeruginosa - CAS 101043-37-2 - CalbiochemSigma AldrichCatalog #475815m
- Pierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
- 4x Protein Loading Buffer
| A | B | |
| Tris-HCl, pH 6.8 | 125 mM | |
| Glycerol | 50% | |
| SDS | 4% | |
| Orange G | 0.2% |
- Acrylamide
- 4x Running buffer
| A | B | |
| Trizma base | 48 g | |
| Glycine | 230.4 g | |
| NaN3 | 20 mL | |
| ddH2O | Diluted to 4 L |
- Running buffer
| A | B | |
| 4x running buffer | 250 mL | |
| ddH2O | 750 mL | |
| 10% SDS | 10 mL |
- Transfer buffer
| A | B | |
| 4x running buffer | 125 mL | |
| ddH2O | 875 mL | |
| 10% SDS | 500 µL | |
| For RABs add 20% Methanol | ||
- Immobilon®-FL PVDF MembraneSigma AldrichCatalog #Ipfl00010
- Chameleon® Duo Pre-stained Protein LadderLI-CORCatalog #928-60000
- Revert™ 700 Total Protein Stain for Western Blot Normalization (250 ml)LicorCatalog #926-11021
- Revert Wash Solution
| A | B | |
| Acetic acid | 6.7% | |
| Methanol | 30% | |
| in ddH2O | ||
- Revert Reversal Solution
| A | B | |
| NaOH | 0.1 M | |
| Methanol | 30% | |
| in ddH2O | ||
- EveryBlot Blocking Buffer 500 mlBIO-RADCatalog #12010020
- Primary antibodies (see Materials and Methods for specific antibodies used)
- Secondary antibodies (see Materials and Methods for specific antibodies used)
Equipment
- ODYSSEY CLx Imaging System (LI-COR)
Equipment
Mini-PROTEAN Tetra Vertical Electrophoresis Cell
NAME
Electrophoresis Cell
TYPE
Bio-Rad
BRAND
1658004
SKU
LINK
Equipment
Mini Trans-Blot Electrophoretic Transfer Cell
NAME
Electrophoretic Transfer Cell
TYPE
Bio-Rad
BRAND
1703930
SKU
LINK
Safety warnings
- Microcystin-LR is an extremely potent hepatotoxin and should be handled with great care.
- Acrylamide is a neurotoxin and should be handled with care.
- Methanol-containing reagents should be handled carefully, as methanol can penetrate single-layer laboratory gloves.
Preparation of cell lysates
Preparation of cell lysates
Quickly wash cells twice with ice-cold PBS. After the second wash, tilt the dish and completely aspirate all residual PBS.
Immediately add ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer. Place cells On ice .
Note
The amount of lysis buffer to use depends on cell confluency / cell number, cell type, and cell culture dish. In most cases, using 100 µL – 150 µL lysis buffer per well of a 6-well plate should result in a protein concentration > 1 µg/µL .
Note
Halt protease and phosphatase inhibitor cocktail and microcystein-LR should be added fresh on the day of use.
Scrape cells off the dish using a cell lifter.
Transfer the cell lysate to an Eppendorf tube On ice .
Leave lysates On ice for 00:20:00 to allow for efficient lysis.
Note
Cell lysates can be snap frozen in liquid nitrogen and stored at -80 °C for future use.
20m
Centrifuge for 00:10:00 at 17000 x g and 4 °C .
10m
Discard pellet and use clarified supernatant to determine protein concentration by BCA assay following the manufacturer’s instructions, performing all measurements in triplicates.
Add 100 µL ß-mercaptoethanol to900 µL of 4x Protein Loading Buffer and mix well.
Add complete 4x Protein Loading Buffer to cell lysates, mix well, and boil for 00:05:00 at 95 °C .
Note
Do not store Protein Loading Buffer with BME for more than two weeks.
5m
SDS-polyacrylamide gel electrophoresis
SDS-polyacrylamide gel electrophoresis
Load samples onto 8% (for LRRK2 protein) to 15% (for PPM1H and Rab proteins) acrylamide gels alongside Chameleon Duo pre-stained protein ladder (LI-COR).
Note
Carefully rinse wells with running buffer before loading cell lysates.
Start electrophoresis at 80 V for 00:20:00 , then increase to 120 V and electrophorese until orange dye runs out.
20m
Protein transfer
Protein transfer
Activate Immobilon-FL PVDF membrane by submerging in methanol for 00:00:30 - 00:01:00 .
1m 30s
Wash in ddH2O and equilibrate in transfer buffer.
Soak sponges in methanol, wash in ddH2O and equilibrate in transfer buffer.
Equilibrate filter paper in transfer buffer.
Assemble blotting sandwich.
Carefully remove any air bubbles between layers using a roller.
Fill transfer tank with ice-cold transfer buffer.
Place transfer system On ice .
Transfer proteins from gel onto PVDV membrane at 100 V for 01:15:00 .
1h 15m
Total protein stain, membrane blocking, and antibody incubation
Total protein stain, membrane blocking, and antibody incubation
1h 41m
1h 41m
After wet-tank transfer, let membrane dry completely for at least 01:00:00 at Room temperature .
1h
Rehydrate membrane for 00:01:00 in 100% methanol, then wash 00:05:00 in 1x TBS.
6m
Incubate for 00:05:00 in Revert total protein stain (LI-COR) while gently shaking at Room temperature .
5m
Wash twice with Revert wash solution, then rinse in ddH2O and image membrane on ODYSSEY CLx imaging system.
Remove Revert total protein stain by incubating membrane in Revert reversal solution for 00:10:00 at Room temperature while gently shaking.
10m
Rinse membrane with ddH2O.
Block membrane for 00:05:00 in Everyblot Blocking Buffer (Bio-Rad) at Room temperature .
5m
Dilute primary antibodies in Everyblot Blocking Buffer and incubate at 4 °C Overnight .
5m
Wash membrane in 1x TBS + 0.1% Tween-20 (TBS-T) at Room temperature (4 washes, 00:05:00 each).
5m
Dilute secondary antibodies 1:20,000 in Everyblot Blocking Buffer and 0.02% SDS.
Note
Incubate membrane in secondary antibodies for 01:00:00 at Room temperature .
Wash membrane in TBS-T at Room temperature (4 washes, 00:05:00 each).
5m
Rinse membrane with TBS (no detergent), then image on ODYSSEY CLx imaging system. Quantify signal intensity using Image Studio Software.