Immunoblotting of macrophages and microglia

Narayana Yadavalli; Shawn M. Ferguson

Jul 07, 2023

Immunoblotting of macrophages and microglia

DOI

dx.doi.org/10.17504/protocols.io.6qpvr41dzgmk/v1

Narayana Yadavalli^1,2,3,4,5,
Shawn M. Ferguson^1,2,3,4,6,5

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

Yale University

DOI: 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

Funders Acknowledgement:

ASAP

Grant ID: 000580

Abstract

This protocol describes the preparation from cell lysate from cultured cells and immunoblotting procedure.

Attachments

mqjwbj3up.docx

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 Temperature4 °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.

ReagentHuman Cathepsin D AntibodyR&D SystemsCatalog #AF1014 

ReagentMouse Monoclonal Antibody to GAPDHEncor BiotechnologyCatalog #MCA-1D4 

ReagentHuman Cathepsin B AntibodyR&D SystemsCatalog #AF953 

ReagentHuman Cathepsin L AntibodyR&D SystemsCatalog #AF952 

ReagentMouse/Rat Cathepsin L AntibodyR&D SystemsCatalog #AF1515 

ReagentHuman Cathepsin C/DPPI AntibodyR&D SystemsCatalog #AF1071 

ReagentHuman Glucosylceramidase/GBA AntibodyR&D SystemsCatalog #MAB7410 

ReagentMITF (D5G7V) Rabbit mAbCell Signaling TechnologyCatalog #12590 

ReagentRecombinant Anti-MiTF antibodyAbcamCatalog #ab303530 

Reagentp70 S6 Kinase AntibodyCell Signaling TechnologyCatalog #9202 

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

ReagentTFEB (D2O7D) Rabbit mAbCell Signaling TechnologyCatalog #37785 

ReagentTFEB Polyclonal antibodyProteintechCatalog #13372-1-AP 

ReagentAnti-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 TemperatureOn ice  .

Aspirate media from cells and rinse cells with PBS TemperatureOn 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 Duration00:05:00  .

Spin down at Centrifigation14000 rpm, 4°C, 00:05:00 .

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 Temperature95 °C   for Duration00:03:00  .

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 Duration01:00:00   in tris-glycine transfer buffer.

Remove nitrocellulose membrane and stain for total protein with ponceau stain.

Wash with milliQ water.

Block membrane with 5% milk in TBST for Duration01:00:00   at TemperatureRoom temperature  .

Incubate membrane with primary antibodies in 2.5% milk in TBST DurationOvernight   at Temperature4 °C  . 
Note
NOTE: Optimal primary antibody incubation time and temperature can be determined empirically for a given primary antibody.

Wash membrane.

Wash membrane for Duration00:10:00   with TBST. (1/3)

10m

Wash membrane for Duration00:10:00   with TBST. (2/3)

10m

Wash membrane for Duration00:10:00   with TBST. (3/3)

10m

Incubate membrane with secondary antibodies conjugated HRP for Duration01:00:00  .

Wash membrane.

Wash membrane for Duration00:05:00   with TBST. (1/3)

Wash membrane for Duration00:05:00   with TBST. (2/3)

Wash membrane for Duration00:05:00   with TBST. (3/3)

Image membranes using a Biorad Chemidco.

	A	B
	Tris-Cl	188 mM
	3% SDS
	30% glycerol
	0.01% bromophenol blue
	15% β-mercaptoethanol

	A	B
	tris base	25 mM
	glycine	192 mM
	0.1% SDS in milliQ milliQ water

	A	B
	NaCl	150 mM
	Tris	10 mM
	EDTA	0.5 mM
	0.5% NP40

	A	B
	Tris-Cl	50 mM
	NaCl	150 mM

Public workspaceImmunoblotting of macrophages and microglia

Immunoblotting of macrophages and microglia