Mar 21, 2020

Public workspaceBrooks Lab Western Blotting Protocol

Genome Biology
DOI
dx.doi.org/10.17504/protocols.io.bcsmiwc6
  • Brooks Lab University of California1,
  • Eva Robinson1,
  • Alison Tang1
  • 1University of California, Santa Cruz
  • BrooksLabUCSC
Alison Tang
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bcsmiwc6
External link: https://doi.org/10.1186/s13059-024-03301-y
Protocol Citation: Brooks Lab University of California, Eva Robinson, Alison Tang 2020. Brooks Lab Western Blotting Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bcsmiwc6
Manuscript citation:
Tang AD, Felton C, Hrabeta-Robinson E, Volden R, Vollmers C, Brooks AN (2024) Detecting haplotype-specific transcript variation in long reads with FLAIR2. Genome Biology 25(). doi: 10.1186/s13059-024-03301-y
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: February 21, 2020
Last Modified: March 21, 2020
Protocol Integer ID: 33325
Keywords: western blot, western blotting, protein expression, proteins,
Abstract
This is a Western Blotting Protocol for Brooks Lab, Department of Biomolecular Engineering, University of California, Santa Cruz.
Attachments
Icon for Brooks_Lab_Western_Protocol.pdf
Brooks_Lab_Western_P...
109KB
Materials
MATERIALS
ReagentPierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
Reagent4–15% Mini-PROTEAN® TGX™ Precast Protein Gels, 10-well, 30 µl Bio-Rad LaboratoriesCatalog #4561083
ReagentPierce RIPA Lysis and Extraction BufferThermo Fisher ScientificCatalog #P189900
ReagentcOmplete™ Mini Protease Inhibitor Cocktail (Roche)Merck MilliporeSigma (Sigma-Aldrich)Catalog #04693124001
Reagent4x Laemmli Sample Buffer Bio-Rad LaboratoriesCatalog #1610747
Reagent2-Mercaptoethanol Bio-Rad LaboratoriesCatalog #1610710
ReagentPrecision Plus Protein™ Dual Color Standards 500 µl Bio-Rad LaboratoriesCatalog #1610374
ReagentPrecision Plus Protein™ Dual Color Standards 500 µl Bio-Rad LaboratoriesCatalog #1610374
ReagentWestern Blot Box (black size 8.9 cm × 6.5 cm × 2.5 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z742099-5EA
Reagentβ-Actin Antibody (C4)Santa Cruz BiotechnologyCatalog #sc-47778
ReagentSuperSignal™ West Pico PLUS Chemiluminescent SubstrateThermo Fisher ScientificCatalog #34577
Note: You can use Pierce BCA Protein Assay Kit #23225 or #23227


Additional Reagents required:

  • 100 mM Tris-Cl, pH 8.0
  • 300 mM NaCl
  • 10% NP-40 in ddH2O
  • 10% Na-deoxycholate in ddH2O (light sensitive!)
  • 10% SDS
  • 10x PBS
  • Tween-20
  • 190 proof ethanol
  • 10x TGX running buffer
  • Safeway brand non-fat milk pouch
  • PBST

  • BioRad Transblot kit

  • LoBind microcentrifuge tubes Protein, 1.5 ml


Additional Equipment

  • Sonicator or Bioruptor
  • Microplate reader
  • 200 μl pipettor
  • Varioskan
  • BioRad Gel Doc EZ Gel Documentation System
  • BioRad ChemiDoc XRS+
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and environmental hazards.
RIPA Lysis Buffer
RIPA Lysis Buffer
Make Amount100 mL RIPA w/o protease inhibitor , cover, and freeze in 10 ml aliquots at Temperature-20 °C .

OR

Use pre-made Pierce RIPA buffer, 100 ml, which is aliquoted in Amount10 mL aliquots and stored in Temperature-20 °C
Before use, thaw and add 1 tablet of protease inhibitor (PI) per 10 ml aliquot. (Roche cOmplete™, Mini Protease Inhibitor Cocktail).
Harvesting Cells and Preparing Lysate
Harvesting Cells and Preparing Lysate

Note
Always keep everything on ice, unless otherwise indicated.

Wash confluent 10 cm plate of cells 2X in ice cold PBS.
Add Amount1 mL cold RIPA with PI to cells, and scrape the cells to remove them from the dish. Transfer to a pre-chilled 2 ml tube TemperatureOn ice .

Incubate TemperatureOn ice Duration00:00:20 , with periodic vortexing.

Incubation
Pellet the insoluble material by spinning at max speed in refrigerated (Temperature4 °C ) microcentrifuge Duration00:00:10 .

Centrifigation
Transfer supernatant to a clean LoBind Protein 1.5 ml tube as lysate. To avoid multiple freeze-thaw cycles, make aliquots (generally Amount200 µL each).
Note
Additional Sonicator or BioEruptor sonication step often needed here for more complete nucleic acid removal. See below.

Store lysates aliquots at Temperature-80 °C .
Sonication of Lysate to break up nucleic acids
Sonication of Lysate to break up nucleic acids
Place samples in a 1.5 ml LoBind Protein microfuge tube and prepare a volume balance tube.
Bring a timer, ear cuffs, TemperatureOn ice samples, and balance to second floor to Kamakaka Lab to get the plastic tube adaptor (in back of second drawer in 2nd bay from the back of the lab.)

Sonicator is in cold room down the hall. Put the outflow tube up on the shelf and fill glass reservoir with water to top of white line.
Sonicator settings: timer on hold, use max setting (10), constant, then flip on.
Hold tubes in the adaptor in the sonicating water bath for Duration00:00:30 .
Place tubes TemperatureOn ice to cool for Duration00:01:00 .

Repeat the 30’ sonication (= total of two rounds on max at Duration00:00:30 .)

BCA Assay for Protein Concentration Determination
BCA Assay for Protein Concentration Determination
Pierce BCA Protein Assay Kit #23225 or #23227, Thermo Sci with 2 mg/ml BSA standard.

Use instructions from kit.
Equilibrate reagents, samples, and standards to TemperatureRoom temperature . Use the Microplate reader instructions.
Prepare a dilution series of BSA in working range of Concentration20 μg/ml – Concentration2000 μg/ml from 1 glass vial of stock 2 mg/ml BSA in the kit for the standard curve:
VialRIPA buffer, μl2 mg/ml BSA or standard, μlFinal con μg/ml
A0300 of stock2000
B125375 of stock1500
C325325 of stock1000
D175175 of B750
E325325 of C500
F325325 of E250
G325325 of F125
H400100 of G25
I40000

Prepare fresh working reagent 50:1 of A:B, enough for all standards, unknowns, and replicates of the unknowns (should have n=3).
Add Amount25 µL of unknown or standard to 96-well plate well followed by Amount200 µL working reagent using 200 μl pipettor and gently mix avoiding spillover.
Cover plate with foil and incubate Temperature37 °C for Duration00:30:00 in shaking incubator, gently shaking.
Incubation
Cool to TemperatureRoom temperature .

Measure Absorbance on the VarioSkan at 562 nm.
Determine protein concentration of your unknown from the standard curve (the Skanit software will plot the curve and the unknowns on it if you edit the standards in the plate layout to include the concentrations above in the table.)
Analyze
Export report as an excel doc.
SDS PAGE
SDS PAGE
Prepare the following solutions for SDS-PAGE, Transfer, and Antibody Incubations:

Running Buffer (Tris-Glycine)
100 ml10x TGX running buffer
900 mldd water

Transfer Buffer
200ml5x Biorad Transfer Buffer
200ml 190 proof ethanol
600mldd water

PBST
50 ml10x PBS
0.5 mlTween-20
bring vol to 500 ml with dd water

5 % Milk Block (prepare immediately before use!)
5 gSafeway brand non-fat milk pouch
100 mlPBST

SDS PAGE: Denaturing samples in Laemmli reducing buffer
SDS PAGE: Denaturing samples in Laemmli reducing buffer
In a fume hood, Add Amount100 µL 2-mercaptoethanol to Amount900 µL 4x Laemmli sample buffer to make MLB.
Dilute samples with MLB at a ratio of 3 parts sample to 1 part MLB. So for Amount30 µL final volume, add Amount7.5 µL MLB to Amount22.5 µL sample in RIPA in 1.5 ml LoBind safety lock tube.
Denature at Temperature95 °C for Duration00:05:00 .
Transfer to ice.
SDS PAGE: Loading and Running Gel
SDS PAGE: Loading and Running Gel
Remove gel from package, take off green strip at the bottom, and rinse the wells three times with about Amount1 mL running buffer (use 1 ml pipettor).
Wash
Place gel, tall plate facing out in outer side of a holder and the reservoir block at the other to make a running buffer reservoir. Put the other gel holder in place to take up space.
Fill the chamber to the 2-gel mark. Make sure there are no leaks before testing the circuit. Test the circuit by checking for bubbles after turning on power to 70 volts.
Using a 20 ul conte-tipped pipettor, load Amount30 µL of the samples in MLB onto gel, as well as Amount10 µL of Biorad Precision Plus protein ladder Dual Color.
Run at 100 – 120V, depending on desired resolution, until adequate separation of ladder lanes in sizes regions of interest.
Photograph the gel after the run with your phone to help keep track of orientation.
Semi-dry Transfer to PVDF Membrane
Semi-dry Transfer to PVDF Membrane
Pre-soak blotting stacks in 1X transfer buffer made according to directions on the bottle.
Pre-wet PVDF membrane from Biorad Transblot kit in 190 proof ethanol, then in transfer buffer.
Layer into Transblot drawer (stack, membrane, gel, second stack) before rolling out bubbles gently with conical tube
Run the transfer on Biorad setting, standard Mininigel TGX, 25mA, 25V, Duration00:03:00 .

While running, pour 5% milk block in 1X PBST directly into cleaned small black Western blot box.
Incubate transferred blot for Duration01:00:00 at TemperatureRoom temperature in 5% milk block in 1X PBST.
Incubation
Photograph the blot after the transfer to help keep track of orientation.
Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding
Antibody Incubations and ECL visualization of target protein bands: Primary and secondary antibody binding
Primary and secondary antibody binding

  • β-Actin (C4): sc-47778
  • ECL SuperSignal West Pico Chemiluminescent Substrate

Use 1:500 Dilutions for primary antibodies, in 5% milk/PBST
Note
Note: Primary antibody (unconjugated with HRP) can be re-used 3x if kept frozen.

Drain milk block from blots and add primary Ab (in milk block).
Incubate in cold room DurationOvernight covered and shaking on orbital shaker, covered with plastic.
Incubation
The next day, wash blot 3x/Duration00:15:00 in PBST.
Wash
Freshly prepare secondary antibody at 1:10,000 dilution (Amount2 µL to Amount20 mL 5% milk/PBST ).
Incubate in secondary antibody for Duration01:00:00 , TemperatureRoom temperature .
Incubation
Discard secondary and wash blot 3x/Duration00:05:00 in 1xPBST.
Wash
Discard secondary and in the same box or moving the blot to a new box, add ECL working solution (Amount1 mL of both reagents mixed in a Amount15 mL conical and applied to blot.)
Incubate in ECL for Duration00:05:00 , shaking occasionally by hand, covered.
Incubation
Drain blot and place between plastic sheets for imaging using Biorad GelDoc, as below. After imaging, the blot can be probed for actin as a protein loading control without stripping the blot, using anti-actin-HRP as described below.
Antibody Incubations and ECL visualization of target protein bands: Actin staining with anti-actin-HRP primary
Antibody Incubations and ECL visualization of target protein bands: Actin staining with anti-actin-HRP primary
Actin staining with anti-actin-HRP primary

Wash blot 3x with 1X PBST < Duration00:10:00 .
Wash
Incubate Duration00:20:00 , TemperatureRoom temperature in the primary actin antibody 1:500 freshly made dilution in 5% Milk block.
Incubation
Wash 2 – 3x in 1x PBST Duration00:05:00 each.
Wash
Incubate in ECL as above (step 51) and image.
Incubation
Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab)
Antibody Incubations and ECL visualization of target protein bands: Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab)
Imaging Blot on BioRad ChemiDoc XRS+ (Vollmers lab)

Place blot in between sheet protector plastic and transport to imager in cassette to keep dark.
Place blot on white screen.
Place screen into chamber drawer.
Select new protocol, then blot, then chem hi sensitivity and position blot under live focus setting.
Under Applications, select chemidoc hi sensitivity to photograph target bands. Note the image size, the gain (2X), and bin (2x2) to make sure the image size is the same when the protein standards are photographed in order to be able to merge the images.
Under Live Acquire, select acquisition settings: 1, 600, for range and total =100 acquisitions (the 100 can be lowered for longer exposure), starting at 0.25 start time for high expression target.
Freeze and save the image of your choice as it comes up.
Without moving the blot, take a picture to visualize the protein standards by selecting Custom under Applications, then create an Epi illumination protocol with the same gain and binning as used for photographing the bands (double check the image size prediction under the options…)
Merge the target bands and the protein standards pictures in Image Lab software using Image tools.
Note
Note: Imaged blots can be stored at 4ºC in PBST for stripping and re-probing. Some folks also store them frozen in -20ºC flat in plastic bag.