Feb 07, 2018
Western Blot Protocol
- 1University of Virginia, Charlottesville
- Boster Bio
Protocol Citation: Cj Xia 2018. Western Blot Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.k5ycy7w
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 in our group and it is working.
Created: December 05, 2017
Last Modified: March 28, 2018
Protocol Integer ID: 9112
Keywords: western blot, wb, gel electrophoresis, SDS-PAGE, immunoblot
Abstract
Western blotting WB (immunoblotting) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies. WB involves two major
processes: Separation of soluble proteins into discrete bands and transfer of those proteins onto a solid matrix for subsequent analysis by immunological probes.
Under standard denaturing conditions, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate sample proteins based on polypeptide length. The separated proteins are transferred (blotted) onto a membrane matrix of nitrocellulose or PVDF where they are stained with antibody probes specific to target protein antigens. Typically, blots are incubated with antibodies against the antigen of interest followed by a secondary antibody and detection.
Analysis of protein migration and the intensity of chromogenic, chemiluminescent, or fluorescent signals offer protein expression details from cells or tissue homogenates. When
coupled with high-resolution gel electrophoresis, antibodies of strong avidity and specificity to target antigens, and robust signal reporting, Western blotting can detect picogram quantities of protein.
Materials
STEP MATERIALS
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Mammal Cell Protein Extraction ReagentBoster BioCatalog #AR0103
RIPA Lysis BufferBoster BioCatalog #AR0105
Broad Spectrum Protease Inhibitor Cocktail (100x)Boster BioCatalog #AR1182
Mammalian Tissue Protein Extraction ReagentBoster BioCatalog #AR0101
BCA Protein Assay KitBoster BioCatalog #AR0146
Micro BCA Protein Assay KitBoster BioCatalog #AR1110
SDS-PAGE Gel Preparation KitBoster BioCatalog #AR0138
Dual Color Protein Loading Buffer 4XBoster BioCatalog #AR1142
SDS-PAGE Loading Buffer 2XBoster BioCatalog #AR0131
SDS-PAGE Protein Loading Buffer 5X (Reducing)Boster BioCatalog #AR1112
SDS-PAGE Tricine Loading Buffer Boster BioCatalog #AR1143
SDS-PAGE Electrophoresis BufferBoster BioCatalog #AR0139
Coomassie Blue Staining & Destaining SolutionBoster BioCatalog #AR0140
Coomassie Blue Fast Staining SolutionBoster BioCatalog #AR0170
Protein Silver Stain KitBoster BioCatalog #AR0171
Tris-glycine-SDS BufferBoster BioCatalog #AR1149
Fast & Efficient Transfer Pad (12.5 cm x 12.5 cm)Boster BioCatalog #AR0172
Fast & Efficient Transfer Pad (9 cm x 7.5 cm)Boster BioCatalog #AR0173
Imprinted Membrane Fast Reversible Protein Staining ReagentBoster BioCatalog #AR0142
TBS Wash BufferBoster BioCatalog #AR0144
TBS Blocking BufferBoster BioCatalog #AR0143
TBS Wash BufferBoster BioCatalog #AR0144
TBS Wash BufferBoster BioCatalog #AR0144
TBS Blocking BufferBoster BioCatalog #AR0143
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)Boster BioCatalog #EK1001
Enhanced Chemiluminescent (ECL) Detection Kit (Rabbit IgG)Boster BioCatalog #EK1002
Enhanced Chemiluminescent (ECL) Detection Kit (Goat IgG)Boster BioCatalog #EK1003
Enhanced Chemiluminescent (ECL) Detection Kit (Rat IgG)Boster BioCatalog #EK1004
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgM)Boster BioCatalog #EK1005
Hypersensitive ECL Chemiluminiscence SubstrateBoster BioCatalog #AR1170
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-5
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-10
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-25
WB Developing Fixing KitBoster BioCatalog #AR0132
WB Stripping BufferBoster BioCatalog #AR0153
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2020
Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2021
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2022
Western Blotting Rat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2023
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2024
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2025
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Mammal Cell Protein Extraction ReagentBoster BioCatalog #AR0103
RIPA Lysis BufferBoster BioCatalog #AR0105
Broad Spectrum Protease Inhibitor Cocktail (100x)Boster BioCatalog #AR1182
Mammalian Tissue Protein Extraction ReagentBoster BioCatalog #AR0101
BCA Protein Assay KitBoster BioCatalog #AR0146
Micro BCA Protein Assay KitBoster BioCatalog #AR1110
SDS-PAGE Gel Preparation KitBoster BioCatalog #AR0138
Dual Color Protein Loading Buffer 4XBoster BioCatalog #AR1142
SDS-PAGE Loading Buffer 2XBoster BioCatalog #AR0131
SDS-PAGE Protein Loading Buffer 5X (Reducing)Boster BioCatalog #AR1112
SDS-PAGE Tricine Loading Buffer Boster BioCatalog #AR1143
SDS-PAGE Electrophoresis BufferBoster BioCatalog #AR0139
Coomassie Blue Staining & Destaining SolutionBoster BioCatalog #AR0140
Coomassie Blue Fast Staining SolutionBoster BioCatalog #AR0170
Protein Silver Stain KitBoster BioCatalog #AR0171
Tris-glycine-SDS BufferBoster BioCatalog #AR1149
Fast & Efficient Transfer Pad (12.5 cm x 12.5 cm)Boster BioCatalog #AR0172
Fast & Efficient Transfer Pad (9 cm x 7.5 cm)Boster BioCatalog #AR0173
Imprinted Membrane Fast Reversible Protein Staining ReagentBoster BioCatalog #AR0142
TBS Wash BufferBoster BioCatalog #AR0144
TBS Blocking BufferBoster BioCatalog #AR0143
TBS Wash BufferBoster BioCatalog #AR0144
TBS Wash BufferBoster BioCatalog #AR0144
TBS Blocking BufferBoster BioCatalog #AR0143
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)Boster BioCatalog #EK1001
Enhanced Chemiluminescent (ECL) Detection Kit (Rabbit IgG)Boster BioCatalog #EK1002
Enhanced Chemiluminescent (ECL) Detection Kit (Goat IgG)Boster BioCatalog #EK1003
Enhanced Chemiluminescent (ECL) Detection Kit (Rat IgG)Boster BioCatalog #EK1004
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgM)Boster BioCatalog #EK1005
Hypersensitive ECL Chemiluminiscence SubstrateBoster BioCatalog #AR1170
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-5
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-10
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-25
WB Developing Fixing KitBoster BioCatalog #AR0132
WB Stripping BufferBoster BioCatalog #AR0153
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2020
Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2021
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2022
Western Blotting Rat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2023
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2024
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2025
Protocol materials
SDS-PAGE Protein Loading Buffer 5X (Reducing)Boster BioCatalog #AR1112
WB Stripping BufferBoster BioCatalog #AR0153
TBS Wash BufferBoster BioCatalog #AR0144
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-10
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2025
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)Boster BioCatalog #EK1001
Mammalian Tissue Protein Extraction ReagentBoster BioCatalog #AR0101
RIPA Lysis BufferBoster BioCatalog #AR0105
SDS-PAGE Tricine Loading Buffer Boster BioCatalog #AR1143
Protein Silver Stain KitBoster BioCatalog #AR0171
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)Boster BioCatalog #EK1001
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Imprinted Membrane Fast Reversible Protein Staining ReagentBoster BioCatalog #AR0142
TBS Wash BufferBoster BioCatalog #AR0144
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-25
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2024
Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2021
SDS-PAGE Loading Buffer 2XBoster BioCatalog #AR0131
Protein Silver Stain KitBoster BioCatalog #AR0171
Fast & Efficient Transfer Pad (12.5 cm x 12.5 cm)Boster BioCatalog #AR0172
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-5
TBS Wash BufferBoster BioCatalog #AR0144
Enhanced Chemiluminescent (ECL) Detection Kit (Rat IgG)Boster BioCatalog #EK1004
SDS-PAGE Gel Preparation KitBoster BioCatalog #AR0138
Micro BCA Protein Assay KitBoster BioCatalog #AR1110
Enhanced Chemiluminescent (ECL) Detection Kit (Rabbit IgG)Boster BioCatalog #EK1002
SDS-PAGE Gel Preparation KitBoster BioCatalog #AR0138
Dual Color Protein Loading Buffer 4XBoster BioCatalog #AR1142
Enhanced Chemiluminescent (ECL) Detection Kit (Goat IgG)Boster BioCatalog #EK1003
BCA Protein Assay KitBoster BioCatalog #AR0146
SDS-PAGE Electrophoresis BufferBoster BioCatalog #AR0139
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-25
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2022
WB Developing Fixing KitBoster BioCatalog #AR0132
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgM)Boster BioCatalog #EK1005
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2024
Tris-glycine-SDS BufferBoster BioCatalog #AR1149
Enhanced Chemiluminescent (ECL) Detection Kit (Rat IgG)Boster BioCatalog #EK1004
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-5
Tris-glycine-SDS BufferBoster BioCatalog #AR1149
Fast & Efficient Transfer Pad (9 cm x 7.5 cm)Boster BioCatalog #AR0173
Broad Spectrum Protease Inhibitor Cocktail (100x)Boster BioCatalog #AR1182
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2020
Broad Spectrum Protease Inhibitor Cocktail (100x)Boster BioCatalog #AR1182
Mammalian Tissue Protein Extraction ReagentBoster BioCatalog #AR0101
SDS-PAGE Tricine Loading Buffer Boster BioCatalog #AR1143
Coomassie Blue Fast Staining SolutionBoster BioCatalog #AR0170
Dual Color Protein Loading Buffer 4XBoster BioCatalog #AR1142
Coomassie Blue Staining & Destaining SolutionBoster BioCatalog #AR0140
Hypersensitive ECL Chemiluminiscence SubstrateBoster BioCatalog #AR1170
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2022
WB Stripping BufferBoster BioCatalog #AR0153
Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2021
TBS Blocking BufferBoster BioCatalog #AR0143
TBS Blocking BufferBoster BioCatalog #AR0143
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2025
Fast & Efficient Transfer Pad (9 cm x 7.5 cm)Boster BioCatalog #AR0173
Enhanced Chemiluminescent (ECL) Detection Kit (Rabbit IgG)Boster BioCatalog #EK1002
Western Blotting Rat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2023
BCA Protein Assay KitBoster BioCatalog #AR0146
TBS Wash BufferBoster BioCatalog #AR0144
SDS-PAGE Electrophoresis BufferBoster BioCatalog #AR0139
TBS Blocking BufferBoster BioCatalog #AR0143
TBS Blocking BufferBoster BioCatalog #AR0143
Imprinted Membrane Fast Reversible Protein Staining ReagentBoster BioCatalog #AR0142
TBS Wash BufferBoster BioCatalog #AR0144
Enhanced Chemiluminescent (ECL) Detection Kit (Goat IgG)Boster BioCatalog #EK1003
Hypersensitive ECL Chemiluminiscence SubstrateBoster BioCatalog #AR1170
WB Developing Fixing KitBoster BioCatalog #AR0132
SDS-PAGE Protein Loading Buffer 5X (Reducing)Boster BioCatalog #AR1112
Coomassie Blue Staining & Destaining SolutionBoster BioCatalog #AR0140
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2020
Mammal Cell Protein Extraction ReagentBoster BioCatalog #AR0103
SDS-PAGE Loading Buffer 2XBoster BioCatalog #AR0131
Coomassie Blue Fast Staining SolutionBoster BioCatalog #AR0170
Western Blotting Rat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2023
RIPA Lysis BufferBoster BioCatalog #AR0105
TBS Wash BufferBoster BioCatalog #AR0144
Micro BCA Protein Assay KitBoster BioCatalog #AR1110
Fast & Efficient Transfer Pad (12.5 cm x 12.5 cm)Boster BioCatalog #AR0172
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-10
Mammal Cell Protein Extraction ReagentBoster BioCatalog #AR0103
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgM)Boster BioCatalog #EK1005
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Mammal Cell Protein Extraction ReagentBoster BioCatalog #AR0103
RIPA Lysis BufferBoster BioCatalog #AR0105
Broad Spectrum Protease Inhibitor Cocktail (100x)Boster BioCatalog #AR1182
Mammalian Tissue Protein Extraction ReagentBoster BioCatalog #AR0101
BCA Protein Assay KitBoster BioCatalog #AR0146
Micro BCA Protein Assay KitBoster BioCatalog #AR1110
SDS-PAGE Gel Preparation KitBoster BioCatalog #AR0138
Dual Color Protein Loading Buffer 4XBoster BioCatalog #AR1142
SDS-PAGE Loading Buffer 2XBoster BioCatalog #AR0131
SDS-PAGE Protein Loading Buffer 5X (Reducing)Boster BioCatalog #AR1112
SDS-PAGE Tricine Loading Buffer Boster BioCatalog #AR1143
SDS-PAGE Electrophoresis BufferBoster BioCatalog #AR0139
Coomassie Blue Staining & Destaining SolutionBoster BioCatalog #AR0140
Coomassie Blue Fast Staining SolutionBoster BioCatalog #AR0170
Protein Silver Stain KitBoster BioCatalog #AR0171
Tris-glycine-SDS BufferBoster BioCatalog #AR1149
Fast & Efficient Transfer Pad (12.5 cm x 12.5 cm)Boster BioCatalog #AR0172
Fast & Efficient Transfer Pad (9 cm x 7.5 cm)Boster BioCatalog #AR0173
Imprinted Membrane Fast Reversible Protein Staining ReagentBoster BioCatalog #AR0142
TBS Wash BufferBoster BioCatalog #AR0144
TBS Blocking BufferBoster BioCatalog #AR0143
TBS Wash BufferBoster BioCatalog #AR0144
TBS Wash BufferBoster BioCatalog #AR0144
TBS Blocking BufferBoster BioCatalog #AR0143
Enhanced Chemiluminescent (ECL) Detection Kit (Rabbit IgG)Boster BioCatalog #EK1002
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)Boster BioCatalog #EK1001
Enhanced Chemiluminescent (ECL) Detection Kit (Rat IgG)Boster BioCatalog #EK1004
Hypersensitive ECL Chemiluminiscence SubstrateBoster BioCatalog #AR1170
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-5
Enhanced Chemiluminescent (ECL) Detection Kit (Goat IgG)Boster BioCatalog #EK1003
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgM)Boster BioCatalog #EK1005
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-10
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-25
WB Developing Fixing KitBoster BioCatalog #AR0132
WB Stripping BufferBoster BioCatalog #AR0153
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2022
Western Blotting Rat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2023
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2025
Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2021
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2024
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2020
Sample Preparation - Cell Culture Protein Extraction
Sample Preparation - Cell Culture Protein Extraction
Culture cells in the cell culture dish until 80% confluency.
Place the dish on ice and rinse the cells in ice-cold PBS buffer (10 mM Na2HPO4 and 1.8 mM Na2H2PO4 with 0.2% Tween 20; pH 7.4).
Aspirate the PBS and add ice-cold lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl and protease inhibitors).
Scrape adherent cells off the dish using a cold plastic cell scraper or digest the cells with 0.05% trypsin cells with PBS wash. Be as gentle as possible with the cells to avoid inducing cell stress pathways.
Centrifuge the cells at 3,000 rpm at 4°C for 2-3 min.
4 °C
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Gently transfer the cell precipitate into an ice-cold tube.
Add Mammalian Cell Protein Extraction Reagent (AR0103, Boster Bio) into the tube (v/v: 6/1: extraction reagent/cell precipitate) and resuspend vigorously.
Mammal Cell Protein Extraction ReagentBoster BioCatalog #AR0103
If the above solution is murky, sonicate it for 10-15 seconds to break up the proteins.
RIPA Lysis BufferBoster BioCatalog #AR0105
Sonicate and lyse again if the cell solution remains murky.
Centrifuge at ~10,000 rpm at 4°C for 10 minutes. The centrifugation force and time can be varied depending on the cell type.
4 °C
Discard the lipid (at top) and the cell debris (at bottom) by aliquotting the solution in the middle to a fresh tube and keeping the tube at -20°C.
-20 °C
Sample Preparation - Tissue Protein Extraction
Sample Preparation - Tissue Protein Extraction
Place surgically resected tissues in pre-cooled (4°C) normal saline. Make sure to wash off any blood from the tissues.
4 °C
Cut the tissue into small pieces (0.1 g to 1 g each).
Broad Spectrum Protease Inhibitor Cocktail (100x)Boster BioCatalog #AR1182
Mammalian Tissue Protein Extraction ReagentBoster BioCatalog #AR0101
Mince the tissue and place the minced tissue in tissue homogenizer.
Add ice-cold lysis buffer (For a 5 mg piece of tissue, add 300 µL of buffer. Buffer volume should be determined in relation to the amount of tissue present).
Lyse the tissue homogenate on ice for 4-5 hours or at 4°C (high speed) for 5 minutes.
If necessary, sonicate until no tissue chunks remain.
Centrifuge at ~10,000 rpm at 4°C for 10 minutes. The centrifugation force and time can be varied depending on the sample type.
4 °C
Discard the lipid (at top) and the cell debris (at bottom) by aliquotting the solution in the middle to a fresh tube and keeping the tube at -20°C.
-20 °C
Sample Preparation - Protein Quantitation Assay
Sample Preparation - Protein Quantitation Assay
The test tube protocol for our BCA (Bicinchoninic Acid) Protein Assay Kit (AR0146, Boster Bio) is described here. Refer to our Micro BCA Protein Assay Kit datasheet (AR1110, Boster Bio; datasheet) for the microplate protocol where appropriate.
BCA Protein Assay KitBoster BioCatalog #AR0146
Micro BCA Protein Assay KitBoster BioCatalog #AR1110
Sample Preparation - Protein Quantitation Assay - Reagent Preparation
Sample Preparation - Protein Quantitation Assay - Reagent Preparation
Reconstitute the albumin standard ampules (BSA) with 0.9% NaCl or PBS to create a working solution of 2000 µg/mL (Tube A).
Mix thoroughly 50 mL BCA Reagent A with 1 mL BCA Reagent B (i.e. Diluent).
Prepare the diluted BSA standards by mixing the diluent and BSA as follows:
| A | 0 | 600 (From Tube A) | 2000 | |
| B | 100 | 300 (From Tube A) | 1500 | |
| C | 300 | 300 (From Tube A) | 1000 | |
| D | 200 | 200 (From Tube B) | 750 | |
| E | 300 | 300 (From Tube C) | 500 | |
| F | 300 | 300 (From Tube E) | 250 | |
| G | 300 | 300 (From Tube F) | 125 | |
| H | 400 | 100 (From Tube G) | 25 | |
| I (Blank) | 300 | 0 | 0 |
Sample Preparation - Protein Quantitation Assay - Quantitation Assay
Sample Preparation - Protein Quantitation Assay - Quantitation Assay
For the test-tube protocol, the ratio of sample to working range is 1:20.
Pipette 0.1 mL of each standard and unknown sample replicate into an appropriately labeled test tube.
Add 2.0 mL of the working range (WR) to each tube and mix well.
Cover and incubate tubes with one of the protocols:
- Standard Protocol: 37°C for 30 minutes (WR: 25 to 2,000 μg/mL).
- RT Protocol: Room temperature for 2 hours (WR: 25 to 2,000 μg/mL).
- Enhanced Protocol: 60°C for 30 minutes (WR: 5 to 250 μg/mL).
Note
Increasing the incubation time and temperature can increase the net 562 nm absorbance for each test and decrease both the minimum detection level of the reagent and the working range of the protocol. Use a water bath to heat the tubes for either the Standard (37°C incubation) or the Enhanced (60°C incubation) Protocol. Using a forced-air incubator can introduce significant error in color development because of uneven heat transfer.
Cool all tubes to room temperature.
With the spectrophotometer set to 562 nm, “zero” the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples within 10 minutes.
Note
Because the BCA Assay does not reach a true end point, color development will continue even after cooling to room temperature. However, because the rate of color development is low at room temperature, no significant error will be introduced if the 562 nm absorbance measurements of all tubes are made within 10 minutes of each other.
Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm absorbance measurement of all other individual standard and unknown sample replicates.
Prepare a standard curve by plotting the average blank-corrected 562 nm measurement for each BSA standard vs. its concentration in μg/mL. Use the standard curve to determine the protein concentration of each unknown sample.
Electrophoresis - Gel Preparation
Electrophoresis - Gel Preparation
The first step of gel preparation is to determine the gel percentage based on the molecular weight of your protein sample:
| Gel Percentage | 6% | 8% | 10% | 12% | 20% |
If you are not sure of the size of your protein or are looking at proteins of a variety of molecular weights, then a gradient gel may provide the best resolution.
Note
- We recommend using the SDS-PAGE Gel Preparation Kit is available from us (AR0138, Boster Bio). It contains most of the reagents for the gel preparation and can be used to make both SDS-PAGE gel and non-native PAGE gel, respectively.
SDS-PAGE Gel Preparation KitBoster BioCatalog #AR0138
Note
- Many protocols are available for gel preparation. Please refer to the manufacturer’s guidelines for use of specific products.
Note
- Pre-cast gels may also be used instead of making your own gel.
Electrophoresis - Gel Preparation - Resolving Gel Preparation
Electrophoresis - Gel Preparation - Resolving Gel Preparation
Determine volume needed and gently mix the ingredients for the chosen percentage of the resolving gel.
Pour the solution into your gel casting form.
Layer the top of the gel with distilled water.
Wait approximately 30 minutes for the gel to polymerize completely.
Remove the water from the polymerized resolving gel (absorb excess water with paper towel).
Electrophoresis - Gel Preparation - Stacking Gel Preparation
Electrophoresis - Gel Preparation - Stacking Gel Preparation
Determine the volume needed. Gently mix the ingredients and pour the stacking gel on top of the running gel.
Insert sample comb (avoid bubbles).
Allow 30 to 60 minutes for complete gel polymerization.
Electrophoresis - Pre-electrophoresis Sample Preparation
Electrophoresis - Pre-electrophoresis Sample Preparation
Mix the extracted protein sample with 4X Dual Color Protein Loading Buffer (AR1142, Boster Bio) at 3:1 ratio (i.e. add 300µg sample to 100µL Loading Buffer).
Dual Color Protein Loading Buffer 4XBoster BioCatalog #AR1142
Note
Dual Color Protein Loading Buffer is designed to prevent protein degradation during sample heating prior to electrophoresis and is able to work against pH changes during the SDS-PAGE run. Many proteins are sensitive to pH changes that result from temperature fluctuations of Tris buffers during electrophoresis.
It contains 2 tracking dyes: blue (Bromophenol Blue) for tracking the progress of electrophoresis and pink (Pyronin Y) for monitoring protein transfer to the membrane. Refer to the datasheet on our website for more information.
You may also use one of the following reagents/methods instead of the Dual Color Protein Loading Buffer:
- 2X SDS-PAGE Protein Loading Buffer (AR0131, Boster Bio) at 1:1 ratio (i.e. add 100 µg sample to 100 µL Loading Buffer)
- 5X SDS-PAGE Protein Loading Buffer (AR1112, Boster Bio) at 4:1 ratio (i.e. add 400 µg sample to 100 µL Loading Buffer)
- 2X SDS-PAGE Tricine Protein Loading Buffer (AR1143, Boster Bio) at 1:1 ratio (i.e. add 100 µg sample to 100 µL Loading Buffer) if detecting protein with MW < 10 kDa
- Laemmli 2X Buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl; pH 6.8) at 1:1 ratio (i.e. add 100 µg sample to 100 µL Loading Buffer)
SDS-PAGE Loading Buffer 2XBoster BioCatalog #AR0131
SDS-PAGE Protein Loading Buffer 5X (Reducing)Boster BioCatalog #AR1112
SDS-PAGE Tricine Loading Buffer Boster BioCatalog #AR1143
Denature the sample/loading buffer mixture in a 100°C water bath for 5 minutes (or follow the manufacturer instructions). Alternatively, the mixture can be stored in aliquots at -20°C for several months or at 4°C for 1-2 weeks before use.
Electrophoresis - Loading Samples and Running Electrophoresis
Electrophoresis - Loading Samples and Running Electrophoresis
Place the gel in the electrophoresis apparatus.
Fill both buffer chambers with SDS-PAGE Electrophoresis Buffer (25 mMTris base, 190 mM glycine and 0.1% SDS; pH 8.3); We recommend using our buffer (AR0139, Boster Bio).
SDS-PAGE Electrophoresis BufferBoster BioCatalog #AR0139
Carefully remove the well-creating comb from the gel and rinse wells with the electrophoresis buffer.
Pipette your samples into the wells quickly to prevent possible sample diffusion inside the well (For a well with maximum 30 µL, load 20 to 25 µL of 1 µg/µL sample per well).
Pipette 10 µL of appropriate controls and/or molecular weight standards in separate well(s).
Connect the anode and cathode of the electrophoresis appropriately.
Turn on the power to run electrophoresis at 100V/130V* until the bromophenol blue dye reaches the gel bottom (This can take 1.5 to 3 hours). You should observe fine bubbles from the gel apparatus bottom as this observation indicates sufficient electric current is generated.
Note
- In a discontinuous system, the electrophoresis voltage for stacking gel is lower than that for resolving gel to ensure that proteins are concentrated on the same level before running into the resolving gel.
Note
* The applied voltage should be adjusted according to the gel thickness, the power supply used, and the resolution desired.
Turn off the power when the protein samples have finished migrating in the gel.
Protein Transfer (To Membrane) - Gel Staining (Optional)
Protein Transfer (To Membrane) - Gel Staining (Optional)
After electrophoresis, we recommend using one of our gel staining solutions to determine if the electrophoretic separation works. Refer to the datasheet(s) on our website for more information.
Note
Stained gel cannot be used in the subsequent protein transfer procedure.
Coomassie Blue Staining & Destaining SolutionBoster BioCatalog #AR0140
Coomassie Blue Fast Staining SolutionBoster BioCatalog #AR0170
Protein Silver Stain KitBoster BioCatalog #AR0171
Protein Transfer (To Membrane) - Wet Transfer - Blotting Membrane Preparation
Protein Transfer (To Membrane) - Wet Transfer - Blotting Membrane Preparation
Cut the blotting membrane (NC or PVDF) according to the size of your gel.
Note
Tips: Cut a good supply of membranes in advance! Store in a cool, dry place.
Carefully mark the membrane orientation by cutting a corner or marking it with a pencil.
Soak the membrane in methanol for 1 minute.
Immerse the membrane in 5 minutes with 1X transfer buffer (25 mMTris base, 190 mM glycine and 20% methanol; pH 8.3) (AR1149, Boster Bio) and rock the membrane gently until it sinks and the water no longer beads up on the surface.
Tris-glycine-SDS BufferBoster BioCatalog #AR1149
Protein Transfer (To Membrane) - Wet Transfer - Transfer Cassette
Protein Transfer (To Membrane) - Wet Transfer - Transfer Cassette
Based on a sandwich model, install the electric transfer cassette in the following order:
Foam Pad → Filter Paper → Gel → Membrane→ Filter Paper→ Foam Pad
We recommend using one of the transfer pads from Boster:
Fast & Efficient Transfer Pad (12.5 cm x 12.5 cm)Boster BioCatalog #AR0172
Fast & Efficient Transfer Pad (9 cm x 7.5 cm)Boster BioCatalog #AR0173
Soak two filter papers in a separate container with the same transfer buffer.
Crack open the transfer cassette with a spatula and make sure to loosen the cassette hold all the way around before carefully pulling apart the two halves.
Note
Before doing anything with the gel, such as cutting it, pay careful attention to the location of lane #1.
Cut the gel according to the size of the membrane with a razor blade and then cut the corner on the side of the gel with lane #1.
Immerse the gel in 1X transfer buffer for 15-30 minutes.
Place the gray or black plate of the transfer cassette on a clean surface.
Place one pre-wetted foam pad on the gray side of the cassette.
Place a moistened sheet of filter paper on the foam pad.
Carefully peel the gel off of the remaining half of the gel cassette and place it on the filter paper.
Note
Moisten the gel with transfer buffer and use a serologically clean pipette or a Falcon tube, as if it were a rolling pin, to roll air bubbles out of the membrane.
Place the membrane onto the gel with the corners matched up.
Note
Once the membrane contacts the gel, it should not be moved or “ghost bands” can result.
Complete the sandwich by placing a piece of filter paper onto the membrane.
Add the second foam pad on top of the filter paper.
Lock the transfer cassette firmly with the white latch.
Note
Be careful not to move the gel and the filter paper sandwich. Make sure that the foam pads, filter papers, and membrane are thoroughly immersed in the transfer buffer.
Protein Transfer (To Membrane) - Wet Transfer - Protein Transfer Run
Protein Transfer (To Membrane) - Wet Transfer - Protein Transfer Run
Fill transfer tank with an adequate amount of 1X transfer buffer.
Firmly insert transfer cassette into the slot of the transfer apparatus.
Place the lid on top of the transfer tank and make sure the electrodes are lined correctly.
Note
The gel should be closer to the cathode and the membrane should be closer to the anode. Negatively charged proteins will migrate towards the anode.
Set the power source to constant voltage and operate at 25 V for 30 minutes.
Note
- The transfer can be completed overnight at a lower voltage, for example, 10 V.
Note
Transfer time and voltage should be optimized according to the gel concentration. Higher gel concentration requires additional time.
Check the protein transfer efficiency by membrane staining:
Place the membrane in Ponceau S staining (0.2% w/v Ponceau S; 5% glacial acetic acid) or our Imprinted Membrane Fast Reversible Protein Staining Reagent (AR0142, Boster Bio) for 5-10 minutes. A visible red band will appear. The membrane may be de-stained completely by repeatedly washing in wash buffer.
Imprinted Membrane Fast Reversible Protein Staining ReagentBoster BioCatalog #AR0142
Membrane Blocking
Membrane Blocking
Rinse the blotting membrane 3X using TBS Wash Buffer (20 mMTris, pH 7.5; 150 mMNaCl; 0.05% Tween 20) (AR0144, Boster Bio) at room temperature for 10 minutes each time.
TBS Wash BufferBoster BioCatalog #AR0144
After rinsing, immerse the blotting membrane in TBS Blocking Buffer (5% non-fat dry milk in buffer of 20 mMTris, pH 7.5; 150 mMNaCl) (AR0143, Boster Bio) and incubate for 1.5 - 2 hours at room temperature (or overnight 4°C) with shaking.
Note
Alternatively, buffer containing non-fat dried milk, gelatin, or BSA can be used. For use with biotin systems or detection of phosphoproteins, non-fat dried milk is not recommended.
TBS Blocking BufferBoster BioCatalog #AR0143
Antibody Incubation
Antibody Incubation
After blocking, the membrane is incubated with a primary antibody (that binds to the target protein) followed by an HRP-conjugated or AP-conjugated secondary antibody.
Dilute the primary antibody with the TBS Wash Buffer (AR0144, Boster Bio). Follow the antibody protocol from the manufacturer for optimal dilution.
TBS Wash BufferBoster BioCatalog #AR0144
Incubate the primary antibody and the membrane at 4°C overnight or for 1-2 hours at room temperature. For the best results, incubation time and antibody concentration may need to be optimized.
Wash the membrane 3X with the TBS Wash Buffer (AR0144, Boster Bio) for 10 minutes each to remove unbound antibody.
TBS Wash BufferBoster BioCatalog #AR0144
Dilute the secondary antibody with the TBS Blocking Buffer (AR0143, Boster Bio). Follow the antibody protocol from the manufacturer for optimal dilution.
TBS Blocking BufferBoster BioCatalog #AR0143
Incubate the secondary antibody and the membrane at 4°C overnight or 1-2 hours at room temperature on a shaker.
Wash the membrane 3X with the TBS Wash Buffer for 10 minutes each to remove unbound antibody.
Signal Detection
Signal Detection
In this section, we provide the protocols for the Enhanced Chemiluminescence Detection (ECL) and Colorimetric Detection (DAB) methods. Use the method that fits your preferences and criteria.
Signal Detection - Enhanced Chemiluminescence Detection (ECL) - ECL Substrate Preparation
Signal Detection - Enhanced Chemiluminescence Detection (ECL) - ECL Substrate Preparation
Choose the correct ECL kit† according to the species that the primary antibody is raised.
* Each kit has sufficient reagents for 800 cm2 of membrane.
The ECL kit provides:
1) Chromogenic reagents A and B (20X concentrated; 5 mL)
2) Blocking buffer
3) HRP-conjugated secondary antibody
† Instead of using the ECL kit, one may use one of the following standalone chromogenic reagents A and B from Boster:
** Ready-to-use
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)Boster BioCatalog #EK1001
Enhanced Chemiluminescent (ECL) Detection Kit (Rabbit IgG)Boster BioCatalog #EK1002
Enhanced Chemiluminescent (ECL) Detection Kit (Goat IgG)Boster BioCatalog #EK1003
Enhanced Chemiluminescent (ECL) Detection Kit (Rat IgG)Boster BioCatalog #EK1004
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgM)Boster BioCatalog #EK1005
Hypersensitive ECL Chemiluminiscence SubstrateBoster BioCatalog #AR1170
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-5
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-10
Hypersensitive WB Chemiluminescent SubstrateBoster BioCatalog #CR0001-25
Prepare the ECL substrate solution by mixing the following and use the solution within 2 hours of preparation:
- 50 µL of 20X Chromogenic Reagent A (Luminol & Luminious Enhancer)
- 50 µL of 20X Chromogenic Reagent B (Peroxidase & Stabilizer)
- 1 mL of distilled water
Signal Detection - Enhanced Chemiluminescence Detection (ECL) - Membrane Treatment
Signal Detection - Enhanced Chemiluminescence Detection (ECL) - Membrane Treatment
Thoroughly cover the membrane with the substrate solution (use 1 mL of solution for 10 cm2 of the membrane).
Incubate the membrane at room temperature until bands appear (usually 1-5 minutes; incubation time can be estimated in dark room).
Gently blot the edge of the membrane on a piece of paper to remove excess substrate solution.
Put a clear preservative film or transparent glass paper over the membrane and remove any air bubbles observed.
Signal Detection - Enhanced Chemiluminescence Detection (ECL) - Film Development and Fixing
Signal Detection - Enhanced Chemiluminescence Detection (ECL) - Film Development and Fixing
Develop and fix the film in a dark room immediately using our recommended WB Developing and Fixing Kit (AR0132, Boster Bio). Alternatively, fluorescence CCD scan, digital imager or luminometer can be used.
WB Developing Fixing KitBoster BioCatalog #AR0132
Put the X-ray film over the membrane.
Develop the film by immersing it in developing solution for 10 seconds to 10 minutes. Determine the exposure time required by observing under red light and stop developing once the film achieves the experimental purpose. Multiple exposures may be necessary for the optimal signal to noise ratio.
Wash the film with clean water (to remove the developing solution completely) and stop washing when bands appear.
Immerse the film in fixing solution for 3-5 minutes.
Wash the film with clean water to remove the fixing solution.
Note
- WB Stripping Buffer (AR0153, Boster Bio) is recommended to remove primary and secondary antibodies on the membrane if proteins on the membrane need to be reused.
Note
- Use the control protein levels to normalize the target protein levels.
WB Stripping BufferBoster BioCatalog #AR0153
Signal Detection - Colorimetric Detection - DAB Substrate Preparation (For HRP-conjugated secondary antibodies)
Signal Detection - Colorimetric Detection - DAB Substrate Preparation (For HRP-conjugated secondary antibodies)
Choose the correct DAB kit according to the species that the primary antibody is raised and the desirable color:
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2020
Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2021
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2022
Western Blotting Rat IgG DAB Chromogenic Reagent Kit (Yellow)Boster BioCatalog #SA2023
Western Blotting Mouse IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2024
Western Blotting Rabbit IgG DAB Chromogenic Reagent Kit (Blue)Boster BioCatalog #SA2025
Prepare the DAB substrate solution by mixing the following:
- 50 µL of 40X Chromogenic Reagent A (DAB)
- 50 µL of 40X Chromogenic Reagent B (H2O2)
- 50 µL of 40X Chromogenic Reagent C (TBS Wash Buffer)
- 2 mL of distilled water
Signal Detection - Colorimetric Detection - BCIP/NBT Substrate Preparation (For AP-conjugated secondary antibodies)
Signal Detection - Colorimetric Detection - BCIP/NBT Substrate Preparation (For AP-conjugated secondary antibodies)
Prepare the BCIP/NBT substrate solution by mixing the following:
- 50 µL of 20X Chromogenic Reagent A (BCIP/NBT)
- 50 µL of 20X Chromogenic Reagent B (Tris concentrated buffer, pH 9.4)
- 1 mL of distilled water
Signal Detection - Colorimetric Detection - Membrane Treatment
Signal Detection - Colorimetric Detection - Membrane Treatment
Thoroughly cover the membrane with the substrate solution (use 1 mL of solution for 10 cm2 of the membrane).
Incubate the membrane at room temperature until bands appear (usually 10-30 minutes). Incubation for BCIP/NBT should be done in the dark.
Wash the membrane in distilled water to stop the reaction.
Observe the bands and take pictures.