Jul 31, 2023

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

DOI
dx.doi.org/10.17504/protocols.io.j8nlkk1w1l5r/v1
  • OLIVIA HARDING1,2,
  • Erika L.F. Holzbaur1,2
  • 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
  • Liv
OLIVIA HARDING
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DOI: https://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, ubiquitin in p62, nemo recruitment to damaged mitochondria, biochemical analysis of protein expression, ubiquitin chain, cell lysi, gel electrophoresis for protein analysis, protein analysis, protein expression, nemo colocalization with p62, hela cell, damaged mitochondria, p62 in the soluble fraction, biochemical analysis, ubiquitin, electrophoresi, gel electrophoresi, protein, cell, p62
Funders Acknowledgements:
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
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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 tubesMerck MilliporeSigma (Sigma-Aldrich)Catalog #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)LI-CORCatalog #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 AntibodyLI-CORCatalog #926-32212 and ReagentIRDye® 680RD Donkey anti-Rabbit IgG Secondary AntibodyLI-CORCatalog #926-68073
  • ReagentTrueBlack buffer (Biotium 23013B-1L)BiotiumCatalog #23013B-1L
  • ReagentEveryBlot Blocking Buffer 500 mlBio-Rad LaboratoriesCatalog #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








Troubleshooting
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.


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

5m
Pipetting
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.
Pipetting
Invert samples by hand to mix, and ensure all samples are at the bottom of tubes by briefly centrifuging.
Centrifigation
Mix
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.




Pipetting
For empty lanes, load approx. equal volume of 1X DB.
Pipetting
Gel electrophoresis and immune-blotting: Running
Run samples through stacking gel (85 V, Duration00:20:00 - Duration00:40:00 ).
1h
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
Wash in 1X TBS Duration00:02:00 .

2m
Wash
Stain total protein, Duration00:05:00 , TemperatureRoom temperature , with REVERT 700 Total Protein Stain.

5m
Wash membrane with REVERT wash buffer.

Wash
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
Wash
Rinse in ddWater.
Wash
Gel electrophoresis and immune-blotting: Immuno-labeling
Block membranes in EveryBlot buffer, Duration00:05:00 , TemperatureRoom temperature with rocking.
5m
Incubate in vacuum packs with primary antibodies in EveryBlot DurationOvernight at Temperature4 °C .
Note
See materials and methods for concentrations of antibodies used.

5m
Incubation
Overnight
Wash with TBST.
Wash
Wash with TBST 4x Duration00:05:00 (1/4).
5m
Wash with TBST 4x Duration00:05:00 (2/4).
5m
Wash with TBST 4x Duration00:05:00 (3/4).
5m
Wash with TBST 4x Duration00:05:00 (4/4).
5m
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 .

1h
Incubation
Wash with TBST.

Wash
Wash with TBST 4x Duration00:05:00 (1/4).
5m
Wash with TBST 4x Duration00:05:00 (2/4).
5m
Wash with TBST 4x Duration00:05:00 (3/4).
5m
Wash with TBST 4x Duration00:05:00 (4/4).
5m
Wash 1X with TBS to clear TWEEN.

Wash
Wash 1X with water.
Wash
Image
Imaging
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.