Jul 07, 2023

Immunoblotting of macrophages and microglia

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr41dzgmk/v1
  • Narayana Yadavalli1,2,3,4,5,
  • Shawn M. Ferguson1,2,3,4,6,5
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Berrak Ugur
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr41dzgmk/v1
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. Immunoblotting of macrophages and microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr41dzgmk/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: May 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82458
Keywords: Immunoblot, antibody, tris-glycine, ASAPCRN, preparation from cell lysate, microglia this protocol, cell lysate, microglia, immunoblotting procedure, cultured cell, macrophage, cell
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol describes the preparation from cell lysate from cultured cells and immunoblotting procedure.
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17KB
Materials
Solutions to prepare
RIPA buffer:
AB
NaCl150 mM
Tris10 mM
EDTA0.5 mM
0.5% NP40
supplemented immediately before use with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche).
TBS:
AB
Tris-Cl50 mM
NaCl150 mM
adjust pH to 7.5
TBST: TBS with 0.1% TWEEN-20 (Sigma-Aldrich)
4x Laemmli buffer:
AB
Tris-Cl188 mM
3% SDS
30% glycerol
0.01% bromophenol blue
15% β-mercaptoethanol

Tris-glycine running buffer:
AB
tris base25 mM
glycine192 mM
0.1% SDS in milliQ milliQ water
Tris-glycine transfer buffer:
AB
Tris 25 mM
glycine192 mM
20% methanol in milliQ water
Chill to 4 °C .

Antibody dilutions:

Primary antibodies:

GAPDH,1:10000 dilution (EnCor biotechnology Inc, # MCA-1D4), human specific cathepsin D, 1:4000 dilution (R&D systems #AF1014), mouse specific cathepsin D, 1:5000 dilution (R&D systems #AF1061), human cathepsin B, 1:1000 dilution (R&D systems #AF953), human cathepsin L, 1:10000 dilution (R&D systems # AF952), mouse cathepsin L, 1:2000 dilution (R&D systems # AF1515), human cathepsin C, 1:1000 dilution (R&D systems #AF1071), human GBA, 1:1000 dilution (R&D systems #MAB7410), hLAMP1, 1:2000 dilution (Cell signaling Technology # 9091), mouse LAMP1, 1:2000 dilution (DSHB #ID4B), MITF, 1:1000 dilution (Cell signaling Technology # 125903), MITF, 1:1000 dilution (Abcam #Ab303530), S6K, 1:1000 dilution (Cell signaling Technology #9202 ), S6K-Phospho-T389, 1:1000 dilution (Cell signaling Technology #9205), human TFEB, 1:1000 dilution (Cell signaling Technology #37785), mouse TFEB, 1:1000 dilution (Proteintech #13372-1-AP), TFE3, 1:1000 dilution (Sigma #HPA023881).

Secondary antibodies:

All the HRP tagged secondary antibodies are purchased from Cell signaling Technology and diluted at 1:2000 ratio.

Human Cathepsin D AntibodyR&D SystemsCatalog #AF1014

Mouse Monoclonal Antibody to GAPDHEncor BiotechnologyCatalog #MCA-1D4

Human Cathepsin B AntibodyR&D SystemsCatalog #AF953

Human Cathepsin L AntibodyR&D SystemsCatalog #AF952

Mouse/Rat Cathepsin L AntibodyR&D SystemsCatalog #AF1515

Human Cathepsin C/DPPI AntibodyR&D SystemsCatalog #AF1071

Human Glucosylceramidase/GBA AntibodyR&D SystemsCatalog #MAB7410

MITF (D5G7V) Rabbit mAbCell Signaling TechnologyCatalog #12590

Recombinant Anti-MiTF antibodyAbcamCatalog #ab303530

p70 S6 Kinase AntibodyCell Signaling TechnologyCatalog #9202

Phospho-p70 S6 Kinase (Thr389) AntibodyCell Signaling TechnologyCatalog #9205

TFEB (D2O7D) Rabbit mAbCell Signaling TechnologyCatalog #37785

TFEB Polyclonal antibodyProteintechCatalog #13372-1-AP

Anti-TFE3 antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog #HPA023881

Cell culture and treatments
Supplement RIPA buffer with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche) and chill On ice .

Aspirate media from cells and rinse cells with PBS On ice . Aspirate PBS thoroughly.

Pipette RIPA lysis buffer onto cells and scrape cells using a cell lifter (Corning).
Pipette lysis buffer containing cell mass into Eppendorf tube.
Treat with 25 units of benzinase for 00:05:00 .

5m
Spin down at 14000 rpm, 4°C, 00:05:00 .

5m
Collect supernatant.
Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
Prepare samples at desired concentration and add 4x Laemmli buffer.
Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
4h 48m
Incubate samples at 95 °C for 00:03:00 .

3m
During this incubation, prepare gel apparatus with Mini PROTEAN TGX 4-15% tris-glycine gels (Bio-Rad) and running buffer.
Load samples into gel and run until dye front reaches bottom (250 V).
Remove gel and set up transfer cassette with nitrocellulose membrane.
Transfer at 100 V for 01:00:00 in tris-glycine transfer buffer.

1h
Remove nitrocellulose membrane and stain for total protein with ponceau stain.
Wash with milliQ water.
Block membrane with 5% milk in TBST for 01:00:00 at Room temperature .

1h
Incubate membrane with primary antibodies in 2.5% milk in TBST Overnight at 4 °C .
Note
NOTE: Optimal primary antibody incubation time and temperature can be determined empirically for a given primary antibody.

1h
Wash membrane.
Wash membrane for 00:10:00 with TBST. (1/3)

10m
Wash membrane for 00:10:00 with TBST. (2/3)
10m
Wash membrane for 00:10:00 with TBST. (3/3)
10m
Incubate membrane with secondary antibodies conjugated HRP for 01:00:00 .

1h
Wash membrane.
Wash membrane for 00:05:00 with TBST. (1/3)

5m
Wash membrane for 00:05:00 with TBST. (2/3)
5m
Wash membrane for 00:05:00 with TBST. (3/3)
5m
Image membranes using a Biorad Chemidco.