Cell lysis and gel electrophoresis for protein analysis of HeLa cells

OLIVIA HARDING; Erika L.F. Holzbaur

Jul 31, 2023

Cell lysis and gel electrophoresis for protein analysis of HeLa cells

DOI

dx.doi.org/10.17504/protocols.io.j8nlkk1w1l5r/v1

OLIVIA HARDING^1,2,
Erika L.F. Holzbaur^1,2

¹Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
²Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815

OLIVIA HARDING

Children's Hospital of Philadelphia

DOI: dx.doi.org/10.17504/protocols.io.j8nlkk1w1l5r/v1

Protocol Citation: OLIVIA HARDING, Erika L.F. Holzbaur 2023. Cell lysis and gel electrophoresis for protein analysis of HeLa cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkk1w1l5r/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: June 24, 2022

Last Modified: May 31, 2024

Protocol Integer ID: 65225

Keywords: Cell culture, Cell lysis, Immuno-precipitation, Gel electrophoresis, Western blotting, ASAPCRN

Funders Acknowledgement:

Aligning Science Across Parkinson’s

Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)

Abstract

Here, we present multiple protocols used for biochemical analysis of protein expression and association. First, we used a simple lysis technique to determine the efficiency of an siRNA knockdown. Then, we modified two previously published methods for assaying co-precipitation of p62 and NEMO with magnetic beads conjugated to a GFP-trap molecule. In the first, we pulled down EGFP-NEMO in control or mitochondrial damaged conditions, and in the second, we pulled down EGFP-Ubiquitin in p62-/- cells with expression of wild-type p62 or a dysfunctional mutant. Since p62 is known to form multimers, we used specialized buffers to preserve those putative interactions. We were able to reproduce results published previously by pulling down EGFP-Ubiquitin in p62-expressing cells. However, interestingly, we did not find evidence that NEMO interacts with p62 in the soluble fraction, or via ubiquitin chains generated in basal conditions. These studies demonstrated that NEMO recruitment to damaged mitochondria occurs in specific circumstances, and NEMO colocalization with p62 is also dependent on multiple factors. 

Attachments

470-986.pdf

439KB

Guidelines

 This protocol was developed to analyze protein expression and enrichment in cell culture, including HeLa-M cells and HeLa p62-/- cells.
Option 2 was modified from a protocol used in Turco et al, Molec. Cell, 2019.
Option 3 was modificed from a protocol used in Wurzer et al, eLife, 2015.

Materials

Materials

Reagent1.5 mL capped tubesMillipore SigmaCatalog #EP022364120  
 Cell scrapers
 Liquid nitrogen
ReagentProtein LoBind tubesEppendorfCatalog #022431081  
10% acrylamide gels with desired number of wells (make or purchase)


Reagents:

For all Options:

1X Phosphate buffered saline (PBS)
 ReagentFisher BioReagents™ Bovine Serum Albumin Fraction V Cold-ethanol PrecipitatedFisher ScientificCatalog # BP1605100  

For Option 2:

Reagent10% sodium dodecyl sulfate solution (SDS) Thermo Fisher ScientificCatalog #15553035  

Protease and phosphatase inhibitors

ABC
Leupeptin 1000 X10 mg/mL
DTT1000 X1M
Pepstatin A1000 X1 mg/mL
TAME1000 X10 mg/mL
PMSF100 X100 mM

 
4X Denaturing buffer (DB) (900uL)

AB
SDS4%
Glycerol50%
Tris HCl, pH 6.8125 mM
Orange G0.2% w/v
Betamercaptoethanol (BME)100 uL

Methanol
 4X Running buffer (RB)
1X Tris buffered saline (TBS)
1X TBS with 0.1% Tween ( ReagentTween 20 100% Nonionic DetergentBio-rad LaboratoriesCatalog #1706531 ) (TBST) 


Running Buffer (RB):
AB
4X RB250 mL
Water 750 mL
10% SDS10 mL

Transfer buffer:
AB
4X RB125 mL
Water775 mL
Methanol100 mL
10% SDS500 uL 
Betamercaptoethanol (BME)560 uL

PVDF membranes
ReagentRevert™ 700 Total Protein Stain for Western Blot Normalization (250 ml)LicorCatalog #926-11021  


 REVERT Wash Buffer:

AB
Glacial acetic acid6.7% w/v
Methanol in water30% v/v

REVERT Reversal Buffer:
AB
NaOH0.1 M
Methanol in water30% v/v
 

Desired primary antibodies
 LICOR secondary antibodies such as ReagentIRDye® 800CW Donkey anti-Mouse IgG Secondary AntibodyLicorCatalog #926-32212   and ReagentIRDye® 680RD Donkey anti-Rabbit IgG Secondary AntibodyLicorCatalog #926-68073  
ReagentTrueBlack buffer (Biotium 23013B-1L)BiotiumCatalog #23013B-1L  
ReagentEveryBlot Blocking Buffer 500 mlBIO-RADCatalog #12010020  


Specialized buffers and other reagents:

RIPA buffer (Option 1)

ABCD
ReagentStock concentrationFinal concentrationVolume of stock (for 10 mL)
Tris-HCl (pH 8.0) 1 M50 mM500 uL
EDTA500 mM1 mM20 uL
EGTA 200 mM2 mM100 uL
Triton X-100 10 %1 %1000 uL
DOC5 %0.50 %1000 uL
SDS10 %0.10 %100 uL
NaCl5 M150 mM300 uL
Water--7 mL


Lysis Buffer-A (Option 2):
ABC
Reagent Final conc For 10 mL
1 M HEPES/KOH50 mM  pH 7.5 500 uL
1 M Sorbitol250 mM 2.5 mL
200 mM EGTA 0.5 mM25 uL
1 M Mg-Acetate5 mM50 uL
ddH2O-6.92 mL

PBS with 0.1% TWEEN (PBST) for Option 2


Wash Buffer (Option 3):
ABC
Reagent Final concFor 20 mL
1 M Tris-Cl 20 mM, pH 7.4 400 uL
100% Glycerol 10%  2 mL
5 M NaCl 135 mM 540 uL
ddH2O -17.06 mL


 Lysis Buffer-B (Option 3):
ABC
Reagent Final concFor 5 mL
Master buffer 20 mM, pH 8.04.975 mL
100% NP-40 (IGEPAL) 0.5% 25 uL


ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225 for Option 1 
ReagentChromoTek Spot-Trap® Magnetic Particles M-270ChromoTekCatalog #M-270  


Equipment:

Vacuum apparatus
End-on-end rotating apparatus
Refrigerated centrifuge
Magnetic rack (for GFP-Trap particle precipitation)
Rockers at  TemperatureRoom temperature   and at Temperature4 °C  
Plate reader (such as BioTex Synergy Mx)
Equipment
Odyssey® DLx Imaging System
NAME
Imaging System
TYPE
Licor
BRAND
LI-COR, 9140
SKU
https://www.licor.com/bio/odyssey-dlx/
LINK

ImageStudio software (LI-COR)
Heat source to Temperature95 °C  
Gel electrophoresis apparatus (BIO-RAD)
Membrane transfer apparatus (BIO-RAD)
Excel

Before start

The start point for this protocol is after cells grown on 3.5 cm, or 10cm dishes have been transfected with relevant constructs for Duration18:00:00  - Duration24:00:00   and treated with appropriate small molecules or vehicles.
For 3.5 cm dishes, follow transfection procedures enumerated in imaging protocols.
Chill all reagents TemperatureOn ice  .
Add protease and phosphatase inhibitors to 1X to each lysis buffer immediately before use.

Wash cells

Aspirate media from dishes.

Wash samples quickly x2 with ice cold PBS.
Note
Can stop after washes by scraping cells with the second wash of PBS into 1.5 mL tube, spin down at Centrifigation2400 x g ,  Duration00:03:00  ,  Temperature4 °C  . Aspirate PBS and snap-freeze tubes in liquid nitrogen. If frozen, add respective lysis buffer and inhibitors and let thaw TemperatureOn ice   Duration00:10:00   before proceeding.

Step case

Standard lysis with RIPA
57 steps

Note
 We used this protocol to assess depletion of p62 in HeLa-M cells after siRNA treatment and imaging NEMO recruitment. Samples were collected from Thikness35 mm    imaging dishes.

Add Amount150 µL   RIPA + inhibitors to dish and scrape cells into 1.5 uL tube, OR add buffer to thawed sample and resuspend by pipetting.

Rotate resuspended sample on end-over-end machine at Temperature4 °C   for Duration00:20:00  .

20m

Spin at top speed (Centrifigation17000 x g ), Temperature4 °C  , Duration00:20:00  .

20m

Remove supernatant as Lysis and keep TemperatureOn ice   or store at Temperature-80 °C  .

Measure protein concentration with Pierce BCA assay by adding Amount25 µL   sample or BSA standard to each well in duplicate and Amount200 µL   Reagent A+B. Incubate Temperature37 °C   for Duration00:30:00   then measure absorbance on a plate reader.
Note
It is likely necessary to dilute samples 1:4 or more to measure within the range of the assay.

30m

Add 1/3 volume of 4X DB to remaining Lysis or a measured fraction of sample and heat Temperature95 °C   for Duration00:05:00  .

Proceed to gel electrophoresis.

Gel electrophoresis and immune-blotting

Note
This protocol was developed for use with the LI-COR system for protein detection.

Gel electrophoresis and immune-blotting: Set-up

 Set up electrophoresis cell with 10% gels by manufacturer’s instructions.
 

 Fill cell with RB and flush wells with a plastic transfer pipet.

 Invert samples by hand to mix, and ensure all samples are at the bottom of tubes by briefly centrifuging.

Load wells with equal amounts of protein (Option 1) or equal volumes (Options 2 and 3) and molecular weight standard (Amount4 µL  - Amount5 µL  ).
Note
 We load Amount15 µL  - Amount25 µL   eluate and Amount10 µL   Input.

For empty lanes, load approx. equal volume of 1X DB.

Gel electrophoresis and immune-blotting: Running

Run samples through stacking gel (85 V, Duration00:20:00  - Duration00:40:00  ).
 

 Run samples through 10% gel (125 V, until front has reached bottom of gel, usually ~Duration01:10:00  ).

1h 10m

Gel electrophoresis and immune-blotting: Transfer

Remove gels from electrophoresis cell and construct transfer cassettes with PVDF membranes according to manufacturer’s instructions.

 Place the cassettes in the transfer cell and fill cell with Transfer buffer and icepack.

Place the cell in a basin.

Fill basin with ice around cell.

Run transfer for Duration01:00:00  - Duration01:10:00  , 100 V.

2h 10m

Gel electrophoresis and immune-blotting: Membrane processing and total protein stain

 
Dry membrane between filter paper in the dark for at least Duration00:45:00  .
Note
This is most important for small proteins.
Can be a stopping point for several days.

45m

Rehydrate membrane in MetOH.

Wash in ddWater.

Wash in 1X TBS Duration00:02:00  .

Stain total protein, Duration00:05:00  , TemperatureRoom temperature  , with REVERT 700 Total Protein Stain.

 Wash membrane with REVERT wash buffer.

 Wash membrane 2x Duration00:00:30   with REVERT wash buffer (1/2).

30s

Wash membrane 2x Duration00:00:30   with REVERT wash buffer (2/2).

30s

Image total protein on LICOR.
Note
 Can cut the membrane based on total stain if desired.

Wash off total stain with REVERT Reversal (up to Duration00:10:00  , TemperatureRoom temperature  )

10m

Rinse in ddWater.

Gel electrophoresis and immune-blotting: Immuno-labeling

Block membranes in EveryBlot buffer, Duration00:05:00  , TemperatureRoom temperature   with rocking.
 

Incubate in vacuum packs with primary antibodies in EveryBlot  DurationOvernight   at Temperature4 °C  .
Note
See materials and methods for concentrations of antibodies used.

Wash with TBST.

Wash with TBST 4x Duration00:05:00   (1/4).

Wash with TBST 4x Duration00:05:00   (2/4).

Wash with TBST 4x Duration00:05:00   (3/4).

Wash with TBST 4x Duration00:05:00   (4/4).

Incubate with secondary antibody 1:20,000 in TrueBlack antibody diluent with 0.2% TWEEN and 1:1000 10% SDS for up to Duration01:00:00  .

Wash with TBST.

Wash with TBST 4x Duration00:05:00   (1/4).

Wash with TBST 4x Duration00:05:00   (2/4).

Wash with TBST 4x Duration00:05:00   (3/4).

Wash with TBST 4x Duration00:05:00   (4/4).

Wash 1X with TBS to clear TWEEN.

Wash 1X with water.

Image

Gel electrophoresis and immune-blotting: Quantification

 
For quantification of knockdown (Option 1).

Use ImageStudio software to draw rectangles around total protein in each lane of Total Protein image and subtract
background.

Then add rectangles to outline p62 bands and subtract background.

Transfer intensity measurements to Excel.

Calculate p62 expression relative to total protein for each experiment.

For quantification of p62 enrichment from GFP-NEMO immunoprecipitation.

 Use ImageStudio to add rectangles around input/cytosolic p62 and eluted p62.

Transfer intensity measurements with background subtracted to Excel.

Calculate p62 eluted relative to input amounts.

Calculate elution/input quantity relative one condition.
Note
We found it necessary to perform this secondary normalization due to variability across replicates. In our case, we normalized results from each condition to the EGFP-NEMO + AntA/OligA results.

A	B	C
Leupeptin	1000 X	10 mg/mL
DTT	1000 X	1M
Pepstatin A	1000 X	1 mg/mL
TAME	1000 X	10 mg/mL
PMSF	100 X	100 mM

	A	B
	SDS	4%
	Glycerol	50%
	Tris HCl, pH 6.8	125 mM
	Orange G	0.2% w/v
	Betamercaptoethanol (BME)	100 uL

	A	B
	4X RB	125 mL
	Water	775 mL
	Methanol	100 mL
	10% SDS	500 uL
	Betamercaptoethanol (BME)	560 uL

A	B	C	D
Reagent	Stock concentration	Final concentration	Volume of stock (for 10 mL)
Tris-HCl (pH 8.0)	1 M	50 mM	500 uL
EDTA	500 mM	1 mM	20 uL
EGTA	200 mM	2 mM	100 uL
Triton X-100	10 %	1 %	1000 uL
DOC	5 %	0.50 %	1000 uL
SDS	10 %	0.10 %	100 uL
NaCl	5 M	150 mM	300 uL
Water	-	-	7 mL

A	B	C
Reagent	Final conc	For 10 mL
1 M HEPES/KOH	50 mM pH 7.5	500 uL
1 M Sorbitol	250 mM	2.5 mL
200 mM EGTA	0.5 mM	25 uL
1 M Mg-Acetate	5 mM	50 uL
ddH2O	-	6.92 mL

A	B	C
Reagent	Final conc	For 20 mL
1 M Tris-Cl	20 mM, pH 7.4	400 uL
100% Glycerol	10%	2 mL
5 M NaCl	135 mM	540 uL
ddH2O	-	17.06 mL

A	B	C
Reagent	Final conc	For 5 mL
Master buffer	20 mM, pH 8.0	4.975 mL
100% NP-40 (IGEPAL)	0.5%	25 uL

	A	B
	NaOH	0.1 M
	Methanol in water	30% v/v

Public workspaceCell lysis and gel electrophoresis for protein analysis of HeLa cells

Standard lysis with RIPA57 steps

Cell lysis and gel electrophoresis for protein analysis of HeLa cells

Standard lysis with RIPA
57 steps