Immunoblotting

Will Hancock-Cerutti; Pietro De Camilli

Apr 18, 2022

Immunoblotting

DOI

dx.doi.org/10.17504/protocols.io.bp2l6be9zgqe/v1

Will Hancock-Cerutti^1,2,3,
Pietro De Camilli^1,3

¹Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
²Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
³Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815

William Hancock-Cerutti

DOI: dx.doi.org/10.17504/protocols.io.bp2l6be9zgqe/v1

Protocol Citation: Will Hancock-Cerutti, Pietro De Camilli 2022. Immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6be9zgqe/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: July 08, 2021

Last Modified: May 31, 2024

Protocol Integer ID: 51400

Keywords: Immunoblot, Western blot, Tris-glycine, Tris-acetate, siRNA, cGAMP, HT-DNA, ASAPCRN

Abstract

This protocol describes collection of protein from cultured cells and immunoblotting, including immunoblotting of large proteins using a proprietary tris-acetate buffer system.

Attachments

dxwxbkjz7.pdf

125KB

Materials

Solutions to prepare:

DMEM solution: 
AB
FBS10%
Penicillin100 U/ml
Streptomycin100 mg/ml
L-glutamine2 mM
 
RIPA buffer:
NaCl150 mM
Tris10 mM
EDTA0.5 mM
NP400.50%
Supplemented immediately before use with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche)
TBS: 
AB
Tris-Cl50 mM
NaCl adjust pH to 7.5150 mM
 TBST: TBS with 0.1% TWEEN-20 (Sigma-Aldrich)

3x Laemmli buffer: 
AB
Tris-HCl188 mM
SDS3%
Glycerol30%
Bromophenol blue0.01%
β-mercaptoethanol15%
 Tris-glycine running buffer: 
AB
Tris base25 mM
Glycine192 mM
SDS in milliQ water0.10%
 Tris-glycine transfer buffer: 
AB
Tris25 mM
Glycine192 mM
Methanol in milliQ water20%
Chill to 4˚C
 

Cell culture and treatments

Culture the HeLa-M cells at Temperature37 °C   in 5% CO2 and DMEM containing 10% FBS, Amount100 U/ml   penicillin, Concentration100 mg/mL   streptomycin, and Concentration2 millimolar (mM)   L-glutamine (all from Gibco).

For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.

For experiments using siRNA, transfect 60 pmols of the indicated siRNA using Amount6 µL   Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol. Lyse the cells Duration72:00:00   after siRNA transfection.

For experiments using cGAMP, transfect Amount8 μg/L   of cGAMP using Amount6 µL   Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol.

For experiments using herring testes (HT)-DNA, transfect Amount1 µg   HT-DNA using Amount3 µL   Lipofectamine 2000 (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol.

Cell lysis and sample preparation

40m

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.

Incubate Eppendorf tube TemperatureOn ice   for Duration00:30:00  .

30m

Every 10 minutes, pipette lysis mixture up and down 10 times with a P-200 pipette tip (a total of 3 cycles).
Note
NOTE: Take care not to introduce bubbles.

Centrifuge at Centrifigation15000 x g  for Duration00:10:00   at Temperature4 °C   and collect the post-nuclear supernatant in a new Eppendorf tube.
Note
NOTE: Samples can be snap frozen in liquid nitrogen at this step and stored at -70˚C.

10m

Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).

Prepare samples at desired concentration and add 3x Laemmli buffer.

Gel electrophoresis and immunoblotting (Tris-glycine buffer system)

4h 50m

Incubate samples at Temperature95 °C   for Duration00:05:00  .

During this incubation, prepare gel apparatus with Mini PROTEAN TGX 4-20% trisglycine gels (Bio-Rad) and running buffer.

Load samples into gel and run until dye front reaches bottom (90-120 V).

Remove gel and set up transfer cassette with nitrocellulose membrane.

Transfer at 120 V for Duration01:00:00   at Temperature4 °C   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 Temperature22 °C  .

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 with TBST. Repeat a total of 3 times.

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).

Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 2.5% milk in TBST for Duration01:00:00   at Temperature22 °C  .

Wash membrane with TBST. Repeat a total of 3 times.

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).

Wash membrane with TBS. Repeat a total of 3 times.

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

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

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

Image membranes using a Licor Odyssey Infrared Imager.

Gel electrophoresis and immunoblotting (Tris-acetate buffer system)

20h 55m

For VPS13C immunoblotting, lyse samples and collect the post-nuclear supernatant as above.

Mix post-nuclear supernatant with NuPAGE LDS Sample Buffer and Reducing Agent (Thermofisher) and incubate for Duration00:10:00   at Temperature70 °C  .

10m

During this incubation, prepare gel apparatus with NuPage Tris-Acetate 3-8% gels and NuPage Running Buffer (Thermofisher).

Remove gel and set up transfer cassette with nitrocellulose membrane.

Transfer at 0.05 mA for Duration16:00:00   at Temperature4 °C   in NuPage transfer buffer (Thermofisher).

16h

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 Temperature22 °C  .

Incubate membrane with primary antibodies in 2.5% milk in TBST for Duration02:00:00   at Temperature22 °C  .
Note
NOTE: Optimal primary antibody incubation time and temperature can be determined empirically for a given primary antibody

Wash membrane for with TBST. Repeat a total of 3 times.

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).

Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 2.5% milk in TBST for Duration01:00:00   at Temperature22 °C  .

Wash membrane with TBST. Repeat a total of 3 times.

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).

Wash membrane with TBS. Repeat a total of 3 times.

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

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

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

Image membranes using a Licor Odyssey Infrared Imager.

	A	B
	FBS	10%
	Penicillin	100 U/ml
	Streptomycin	100 mg/ml
	L-glutamine	2 mM

	A	B
	Tris-HCl	188 mM
	SDS	3%
	Glycerol	30%
	Bromophenol blue	0.01%
	β-mercaptoethanol	15%


	NaCl	150 mM
	Tris	10 mM
	EDTA	0.5 mM
	NP40	0.50%

	A	B
	Tris-Cl	50 mM
	NaCl adjust pH to 7.5	150 mM

	A	B
	Tris base	25 mM
	Glycine	192 mM
	SDS in milliQ water	0.10%

	A	B
	Tris	25 mM
	Glycine	192 mM
	Methanol in milliQ water	20%

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Immunoblotting