Oct 08, 2019

Public workspaceWestern Blotting

DOI
dx.doi.org/10.17504/protocols.io.6rmhd46
  • 1Children's Hospital of Philadelphia;
  • 2Center for Childhood Cancer Research
  • Diskin Lab-CHOP
    Tech. support phone: +12674253160 email: conkritek@email.chop.edu
Karina L Conkrite
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DOI: dx.doi.org/10.17504/protocols.io.6rmhd46
Protocol Citation: Karina L Conkrite 2019. Western Blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.6rmhd46
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: August 23, 2019
Last Modified: October 08, 2019
Protocol Integer ID: 27149
Abstract
From growth to isolation and lysis, to quantification, and running the gel, transfer, blotting... the whole bit.
Attachments
Icon for Protein Lysis Buffer.docx
Protein Lysis Buffer...
44KB
Icon for Protein Isolation.docx
Protein Isolation.do...
56KB
Icon for Western Blot Protocol.docx
Western Blot Protoco...
19KB
Guidelines
Keep your proteins cold, on ice at all times.
Be sure of the type of gel needed for the size of the proteins you are interested in.

Materials
STEP MATERIALS
ReagentPierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
Reagent100X Protease/Phosphatase InhibitorCell Signaling TechnologyCatalog #5872
Reagent10X Tris-Glycine bufferBio-Rad LaboratoriesCatalog #1610771
ReagentImmobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010
ReagentWhatman Grade 3MM Chr Cellulose Western Blotting Membranes, GE Healthcare, Grade 3MM Chr Blotting Paper, roll, 10 cm × 100 m,VWR International (Avantor)Catalog #21427-546
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
Protocol materials
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
Reagent100X Protease/Phosphatase InhibitorCell Signaling TechnologyCatalog #5872
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentImmobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentPierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentWhatman Grade 3MM Chr Cellulose Western Blotting Membranes, GE Healthcare, Grade 3MM Chr Blotting Paper, roll, 10 cm × 100 m,VWR International (Avantor)Catalog #21427-546
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
Reagent10X Tris-Glycine bufferBio-Rad LaboratoriesCatalog #1610771
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
Reagent100X Protease/Phosphatase InhibitorCell Signaling TechnologyCatalog #5872
ReagentPierce BCA Protein Assay KitThermo Fisher ScientificCatalog #23225
Reagent10X Tris-Glycine bufferBio-Rad LaboratoriesCatalog #1610771
ReagentImmobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010
ReagentWhatman Grade 3MM Chr Cellulose Western Blotting Membranes, GE Healthcare, Grade 3MM Chr Blotting Paper, roll, 10 cm × 100 m,VWR International (Avantor)Catalog #21427-546
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
Before Starting:
Before Starting:
Before Starting:

Prepare fresh Lysis Buffer for cells. Use 5X stock stored at 4C.
Stock solution consists of

Component1X5X
Tris25 mM125 mM
NaCl150 mM750 mM
EGTA1 mM5 mM
EDTA1 mM5 mM
NaF10 mM50 mM

Remaining ingredients to be added fresh to each sample:
Final concentration
1 mM DTT
1% Triton X-100
1X Protease/Phosphatase Inhibitor (Cell Signalling, Cat #5872)
Per 1 mL 1X buffer:
200 uL 5X stock
100 uL 10% Triton X-100
10 uL 0.1M DTT
10 uL 100X Protease/Phosphatase Inhibitor
680 uL ddH2O

Lysis buffer must be made fresh daily and stored during use on ice.

Grow Cells in culture and treat with whatever desired checmicals/drugs/siRNA/plasmid/etc are desired. Generally, protein is collected 72 hours post treatment. Occasionally, the proteins being monitored may show optimal change at 48 or 96 hours, dependent on cell cycle and protein cycling.
Remove media and wash cells with DPBS.
Collect Cells and either stored as a pellet for lysis later, or lyse directly now,
Reagent100X Protease/Phosphatase InhibitorCell Signaling TechnologyCatalog #5872

Step case

Direct lysis, adherent only
28 steps

If desiring to lyse cells directly in the dish, be certain to remove *all* PBS from the dish so as not to dilute the sample.
Place the dish on ice, add an appropriate amount of lysis buffer to collect the cells. (300 uL/10 cm dish, for example) Use a cell scraper (Corning, 3008) to scrape cells from the surface and displace them. Once detached, cells can be collected in an eppendorf tube for processing.
Lyse cells on ice, 15-30 min
2m
Sonicate samples, (Probe sonicator, Brodeur Lab, setting 6) 5 seconds
Spin samples, 4C, 15', at least 15000g.
Temperature4 °C Centrifigation18000 x g



1m
Without disturbing the pellet, collect supernatant and transfer to clean labeled tube.
Can be stored at -80C. Avoid freeze/thaw cycles. Aliquot if necessary.
Quantification
Quantification
Must quantify protein levels to know how much to load on a gel.
Run Pierce BCA assayGo to


Protocol
BCA Assay for protein quantification
NAME

BCA Assay for protein quantification

CREATED BY
Karina L Conkrite
Ensure your samples are somewhere on the curve. If they are outside the curve, you will need to redo the assay with different dilution of sample:lysis buffer.
Use provided 0.2% BSA for creating a standard curve: Dilute amount necessary for the assay 1:2 using lysis buffer
Label tubes (1.5 mL Eppendorf) for standards.
Final Conc ug/mL Volume 0.1% BSA Volume buffer
Standard (ug) Volume 0.1% BSA (uL) Volume buffer(uL)
0 0 20
2 2 18
4 4 16
6 6 14
8 8 12
10 10 10
15 15 5
20 20 0
Add appropriate amount of Lysis Buffer to all tubes for the standard curve, following chart above.
Label tubes for samples.
Keeping all protein on ice throughout, use 3 uL/ sample + 17 uL Lysis Buffer for a total volume of 20 uL.
Add diluted BSA to standard tubes last, before adding working reagent.
To each sample and all standards, add 1 mL Working Reagent (comprised of [1 mL Reagent A + 20 uL Reagent B] x number of samples + 10% extra for error). Mix well.
Place tubes in 37° C incubator for 30 minutes to react.
3m
Cool samples to room temp.
1m
Aliquot 200 uL from each tube to a well of a clear, 96-well plate. This can be done in triplicate if desired.
Read on BCA program on GloMax plate reader at λ = 562 nm
Use values from BSA standards to create standard curve. Can be done in Excel, Statmost, or GraphPrizm. Seeking R2 value as close to 1 as possible. Ensure all your samples are within the standard curve range. If not, need to be redone.
Use the resultant y=mx+b equation to quantify the amount of protein in each sample. Dividing by 3 will give you your protein concentration per uL.
Prep the samples for running
Prep the samples for running
Select the type and percentage gel you wish to run.
We tend to use Tris-Glycine gels. Percentage depends on your proteins of interest.






Decide how much protein is necessary to run/sample (ug), 15ug? 30 ug?
Still keeping samples on ice, aliquot that amount, plus a suitable amount of 2X sample buffer (stored at -20C) and lysis buffer to make up to a standard volume for all the samples. Keep all samples on ice, Temperature0 °C until the gel is ready to run.
Don't forget to prep your ladders at this time as well.

2X SDS Gel Loading Buffer/ Laemmli Buffer
ComponentEnd Concentrationvol for 4 mLvol for 40 mL
0.5 M Tris-HCl pH 6.8125 mM1 mL10
Glycerol20%0.8 mL8
20% SDS5%0.8 mL8
b-Mercaptoethanol10%0.4 mL4
0.5% Bromophenol Blue0.025%0.2 mL2
H2O0.8 mL8

setting up the gel
setting up the gel
Dependent on the gel type and transfer type you are doing, prep necessary buffers.
For example, if you are running 2 Tris-Glycine gels (Criterion or Novex, both take the same amount of buffer) you will use about 1L TG running buffer.




To make 1L of 1X TG Running buffer:
ComponentVolumeend conc
10X Tris-Glycine buffer, BioRad 1610771100 mL1X
20% SDS, Technova S02935 mL1%
H2O895mL
Reagent10X Tris-Glycine bufferBio-Rad LaboratoriesCatalog #1610771
This buffer may be stored at Temperature4 °C for a time. If using the Criterion set up, buffer may be reused in the tank, but should be fresh in the loading chamber.
If using the SureLock boxes, you will use the entire liter every time.

Also, prep for the transfer. This is very dependent on the size of your proteins of interest. If you are interested in proteins over 150 kDa, you will want to perform a fully wet transfer, possibly overnight in the cold room. In that case, for TG gels, you need to prep the buffer for the transfer, cut the membrane (1) and 3M filter papers (4) necessary to sandwich the transfer.
ReagentImmobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010


ReagentWhatman Grade 3MM Chr Cellulose Western Blotting Membranes, GE Healthcare, Grade 3MM Chr Blotting Paper, roll, 10 cm × 100 m,VWR International (Avantor)Catalog #21427-546







Remove plastic barriers on the bottom of pre-cast gels. If you forget to do this, your proteins will not migrate.
Assemble the gel/box cartridge, locking everything in place. Higher gel casing should be to the outside of the gel box, so you will load from behind. Fill buffer chambers with running buffer and remove combs from gels carefully, so as not to disturb the well edges and displace them.
Using running buffer and a p200 or p1000 pipet, flush the wells to remove the storage buffer.
While you are flushing the wells, heat samples according to the gel type. For example, on a TG gel, samples should be boiled at Temperature95 °C WITH LID LOCKS!!! for Duration00:10:00 use lid locks
Check the directions for the ladder you have selected. Different ladders have different instructions. Some need no boiling at all, some are 2 min, adjust accordingly.



Briefly spin down the tubes to collect samples. Load into gel according to your map using gel loading tips or Rainin p20 tips.

Run the gel
Run the gel
Run the gel according to gel type. For TG gels, we prefer to start slowly (75V) to get through the stacking gel (~20 min). Voltage can then be increased to run faster according to gel type/percentage. Run samples to the bottom of the gel. You can find the optimal voltage/amperage rates for your gels on the info sheets from their supplier.

Novex Gels


Criterion TGX gels

While gels are running, prep your transfer method.
This will depend on your proteins of interest. Low to medium molecular weight proteins (20-150 kDa) can take advantage of the TransBlot Turbo system in the lab.
If you are interested in higher molecular weight proteins, a more traditional wet transfer is called for. Recommended is overnight transfer in the cold room.
IN ALL CASES, prep your necessary buffer ahead of time so it is cold.

Step case

Using TransBlot system
13 steps

transfer occurs from top-down!!
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10016505E.pdf

Stack arrangement in the Trans-Blot Turbo
Using RTA Transfer Kits [1704273 Ready-to-assemble transfer kit includes 40 midi-sized PVDF membranes (8.5 x 13.5 cm), 80 transfer stacks, 2 L 5x transfer buffer, and 2 gel trays for wetting and equilibrating membranes and transfer stacks]:
1. Prepare Trans-Blot Turbo Transfer Buffer: 1 Part 5X trans-blot turbo buffer, 1 part 100% ethanol, 3 parts water. For 1 mini stack, make 100mL. For 2 minis or 1 midi, use 200 mL
2. Wet and equilibrate the membrane and transfer stacks:
PVDF membrane: immerse in 100% methanol or ethanol until the membrane is translucent. Transfer to a soaking tray with 30mL of 1X transfer buffer, submerge the membrane, equilibrate 2-3 minutes
Tranfer stacks: Midi stacks- immerse 2 stacks separated by blue sheet in two soaking trays, each containing 50-70 mL transfer buffer for 2-3 minutes
3. Assemble the sandwich according to figures below.
4. Once assembled, remove excess transfer buffer by inverting the cassette base with the assembled stack carefully held in place. Place the casette lid on and lock into place. Proceed with transfer step.

TransBlot Turbo

Recommended is the preprogrammed "mixed MW" setting, which will run at 1.3A, 25V for 7 minutes. Insert cassettes and hit run!



TRANSFERRING THE GEL TO THE MEMBRANE
TRANSFERRING THE GEL TO THE MEMBRANE
Once you are set up with the appropriate sandwich/buffer/apparatus combo....
Check your current! And transfer...
Check the transfer efficiency
Check the transfer efficiency
Transfer efficiency can be checked by staining and destaining the membrane with Ponceau stain.

0.1% Ponceau S (page 32) in 7% trichloroacetic acid (TCA) for 5 minutes. Rinse the membrane in deionized water to obtain transient staining or 10% acetic acid to obtain permanent staining, and air dry.

If planning to blot immediately, do not allow membrane to dry. Immediately apply blocking solution, or place in TBST until ready to continue.


Blotting
Blotting
After confirming successful transfer, block membrane in 5% milk made in TBST for 1 hour prior to overnight incubation with primary antibody. This should be down with rocking.
For 1 mini gel (9X10cm) 8 mL milk is plenty to block in the Perfect Western boxes.
This will help cut down on non-specific binding of antibodies.

Per 100mL: add 5g powdered milk to 50 mL TBST in bottle with stir-bar. Shake. Add remaining 50 mL TBST and set on stir plate to mix until uniform.

If splitting membrane into sections, cut now.
This is most easily done with a clean scalpel, while the membrane is enclosed in a sheet protector. Recommended to line up the sections with ladder marks on both sides of the membrane and use a ruler for straight edges.
Splitting the membrane is only recommended when you are certain of the antibody's reactivity and patterns. This does allow for staining of multiple targets simultaneously, and the use of less antibody, but can disrupt patterns.

Mix antibody to the specified concentration in the same milk used for blocking buffer (alternately, some antibodies require PBS with serum).
For example, if preparing to stain the entire mini blot at 1:2500 with beta-Actin, you would mix 8 mL of milk with 3.2 uL beta-Actin.
Incubate overnight at Temperature4 °C while rocking


Remove blot from 4C, rinse quickly in TBST,
then wash 3 X 10' in TBST, with rocking.
During the last wash, prepare the secondary antibody
Secondary antibody:
whatever the primary antibody's host species was, you need an antibody against that.
We use HRP conjugated secondaries. So if you incubated against a beta-Actin antibody raised in mouse (ms anti- Beta-Actin) for a secondary you need an HRP conjugated anti-mouse, such as

ReagentAnti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
at a 1:25000 dilution in 5% milk in TBST.
Apply and incubate with rocking, 1 hour

Discard the secondary antibody, rinse blot quickly in TBST,
then wash 3 X 10' in TBST, with rocking.

Develop the blot, using the ECL detection method of choice.
Lowly expressed proteins, recommend
ReagentSuperSignal™ West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCatalog #34095
for other, more abundant proteins, less sensitive substrates may be used.
carefully, without touching anything other than the edges of the blot, remove from TBST and place in a sheet protector. Try to remove all extra TBST.
Apply ECL substrate uniformly to the blot with a pipet, then close the top half of the sheet protector to allow even distribution of the chemical.
Using the Fluor-Chem Q in CTRB 3300, expose the blot and optimize the images. Ensure things are focused prior to exposure and that the iris is open as far as possible. Get the best images you can without overexposing the blot.
Stripping/Reprobe
Stripping/Reprobe
If you need to re-probe the blot for other targets, you can strip the antibodies from the blot and re-probe.
Carefully put the blot in a hybridization tube, with the protein side facing the inside of the tube. Apply 10 mL
ReagentRestore™ Western Blot Stripping BufferCatalog #21059
and incubate in the hyb oven at Temperature37 °C while turning, for 15-20 min



Place blot back in TBST, rinse quickly, then wash 3 x 10' in TBST
Return to step 20 "Blocking" and block, then apply desired primaries at specified dilutions. Follow as before, as many times as desired.