Western Blot Sample Preparation Protocol | Thermo Fisher Scientific

Good sample preparation is critical for successful separation of protein bands in electrophoresis and western blot detection. No single sample preparation method or buffer will work for all sample types due to the diversity of protein samples. However, the following general guidelines and protocols below should provide a good starting point when preparing samples for protein electrophoresis and subsequent western blot analysis.

Search all available Cell Lysis Buffers

Important considerations:

To minimize sample variability, keep sample preparation workflows simple, and use reagents optimized for the specific sample type and target proteins.
Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and lead to degraded proteins. To prevent these negative effects, protease and phosphatase inhibitors should be added to the lysis reagents.
Some buffer components may interfere with the chosen gel electrophoresis chemistry system (e.g., Tris-glycine, Bis-Tris) and cause a variety of artifacts when running the gel. Selecting a gel electrophoresis chemistry that is compatible with the buffer one’s sample is prepared in, is the simplest route. However, if one cannot change the gel electrophoresis chemistry system, one may need to perform sample clean-up to render the sample compatible with the given system.
To avoid under- or overloading samples, determine the protein concentration of each sample prior to electrophoresis with a compatible protein assay.

Cell culture sample preparation
Tissue sample preparation

Preparation of lysate from cell culture for western blot analysis

Materials

Cell lysis buffer (see table below for recommendations)	Sample buffer (loading buffer) (e.g., LDS Sample Buffer, 4x, Cat. No. NP0007)
Protease and phosphatase inhibitor cocktail (e.g., Halt Protease and Phosphatase Inhibitor Cocktail, Cat. No. 78440 or Pierce Protease and Phosphatase Inhibitor Tablet, Cat. No. A32961)	Optional: Sample reducing agent (e.g., NuPAGE Sample Reducing Agent (10X), Cat. No. NP0004)
Cold phosphate-buffer saline (PBS) (e.g., Phosphate-Buffered Saline Buffer Packs, Cat. No. 28372)	Performing native PAGE: Native sample buffer (e.g., Tris-Glycine Native Sample Buffer, Cat. No. LC2673)
Protein assay (e.g., Pierce BCA Protein Assay, Cat. No. 23225)

View or download buffer recipes

Lysis buffer recommendations

Target protein location	Buffer recommended	Description
Whole cell (total protein extraction) (mild lysis buffer to retain protein-protein interactions)	M-PER Mammalian Protein Extraction Reagent	Non-denaturing detergent in 25mM bicine buffer (pH 7.6)
Whole cell (total protein extraction) (protein is membrane bound, nuclear or mitochondria)	RIPA Lysis Buffer	25 mM Tris-HCl pH 7.6 150 mM NaCl 1% NP-40 or Triton X-100 1% sodium deoxycholate 0.1% SDS
Cytoplasmic	NP-40 Cell Lysis Buffer	50 mM Tris, pH 7.4 250 mM NaCl 5 mM EDTA 50 mM NaF 1 mM Na₃VO₄ 1% NP-40

If working with non-mammalian cells, explore the recommended sample type-specific lysis buffers.

Procedure

1. Prepare lysis buffer by adding protease and phosphatase inhibitors. If using Halt Protease and Phosphatase Inhibitor Cocktail 100x, add 10 µL/mL of cocktail directly to the cell lysis buffer. If using Pierce Protease and Phosphatase Inhibitor tablet, dispense tablet from vial and place into 10 mL of lysis buffer and vortex to dissolve. Optional: To inhibit metalloproteases, add EDTA (0.5 M) at 10 µL/mL of lysis buffer.

For adherent cells

2. Place the cell culture dish on ice and carefully remove culture medium and wash the cells with ice-cold PBS.
3. Aspirate PBS and add ice-cold lysis buffer (~1 mL per 10⁷ cells or 100 mm plate; ~0.5 mL for 60 mm plate; ~200-400 µL for 6-well culture plate). Gently shake or swirl for 5 minutes on ice. Alternatively: Cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer in a microcentrifuge tube.
4. Collect the lysate and transfer to a microcentrifuge tube. Centrifuge samples at ~14,000 x g for 15 minutes to pellet the cell debris.
5. Transfer supernatant to a new microcentrifuge tube and discard the pellet.

For suspension-cultured cells

2. Pellet the suspension of cells by centrifugation at 2,500 x g for 10 minutes. Discard the supernatant.
3. Wash the cells once by resuspending the cell pellet in ice-cold PBS. Pellet cells by centrifugation at 2,500 x g for 10 minutes.
4. Add ice-cold lysis buffer (~1 mL per 100 mg or ~100 µL of wet cell pellet). Pipette the mixture up and down to resuspend the pellet.
5. Shake mixture gently for 10 minutes. Remove cell debris by centrifugation at ~14,000 x g for 15 minutes.
6. Transfer supernatant to a new microcentrifuge tube and discard the pellet.

Determination of protein concentration using BCA Protein Assay

7. Prepared diluted albumin (BSA) standards. Alternatively: Use pre-diluted BSA standards.
8. Prepare Working Reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1 Reagent A:B).
9. Pipette 25 µL of each standard or unknown sample replicate into a microplate well (working range = 20–2000 µg/mL).
10. Add 200 µL of the Working Reagent to each well and mix plate thoroughly on a plate shaker for 30 seconds.
11. Cover plate and incubate at 37°C for 30 minutes.
12. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader.
13. Determine protein concentration.

Protocol tip

BCA protein assays have a unique advantage over Bradford assays (Coomassie dye-based assays), as they are compatible with samples that contain up to 5% detergents and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy.

Preparing samples for protein electrophoresis

Denaturing electrophoresis (SDS-PAGE)

Prepare your sample in the appropriate sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
SDS/LDS sample buffer (4X)*	2.5 μL	2.5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

*For tricine gels, it is recommended to use a Tricine SDS sample buffer.

2. Heat samples at 70˚C for 10 minutes.
3. Load samples onto a Bis-Tris, Tris-acetate, or Tris-glycine protein gel.

Protocol tip

Heating the sample at 100°C in SDS-containing buffer can result in proteolysis. We recommend heating samples for denaturing electrophoresis (reduced or nonreduced) at 70°C for 2–10 minutes for optimal results. Do not heat the samples for non-denaturing (native) electrophoresis.

Non-denaturing electrophoresis (Native-PAGE)

Prepare your sample in the appropriate sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
Native sample buffer (2X)	5 μL	5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

2. Load samples onto Tris-glycine or Tris-acetate protein gel.

Preparation of lysate from tissues for western blot analysis

Materials

Cell lysis buffer (see table below for recommendations)	Sample buffer (loading buffer) (e.g., LDS Sample Buffer, 4x, Cat. No. NP0007)
Protease and phosphatase inhibitor cocktail (e.g., Halt Protease and Phosphatase Inhibitor Cocktail, Cat. No. 78440 or Pierce Protease and Phosphatase Inhibitor Tablet, Cat. No. A32961)	Optional: Sample reducing agent (e.g., NuPAGE Sample Reducing Agent (10X), Cat. No. NP0004)
Cold phosphate-buffer saline (PBS) (e.g., Phosphate-Buffered Saline Buffer Packs, Cat. No. 28372)	Performing native PAGE: Native sample buffer (e.g., Tris-Glycine Native Sample Buffer, Cat. No. LC2673)
Protein assay (e.g., Pierce BCA Protein Assay, Cat. No. 23225)

View or download buffer recipes

Lysis buffer recommendations

Target protein location	Buffer recommended	Description
Whole cell (total protein extraction)	T-PER Tissue Protein Extraction Reagent (mild lysis buffer to retain protein-protein interactions and minimize denaturation)	Non-denaturing detergent in 25mM bicine, 150mM sodium chloride (pH 7.6)
Whole cell (total protein extraction) (protein is membrane-bound, nuclear, or mitochondrial)	RIPA Lysis Buffer (contains iconic detergents to assist with bringing proteins into solution)	25 mM Tris-HCl pH 7.6 150 mM NaCl 1% NP-40 or Triton X-100 1% sodium deoxycholate 0.1% SDS

Procedure

1. Prepare lysis buffer by adding protease and phosphatase inhibitors. If using Halt Protease and Phosphatase Inhibitor Cocktail 100x, add 10 µL/mL of cocktail directly to the cell lysis buffer. If using Pierce Protease and Phosphatase Inhibitor tablet, dispense tablet from vial and place into 10 mL of lysis buffer and vortex to dissolve. Optional: To inhibit metalloproteases, add EDTA (0.5 M) at 10 µL/mL of lysis buffer.
2. Dissect the tissue of interest on ice and weigh samples. Use a ratio of ~50 mg tissue to 1,000 µL of ice-cold lysis buffer. Use a smaller volume of lysis buffer if a more concentrated protein extract is required.
3. Add the appropriate amount of ice-cold lysis buffer to the tissue sample and homogenize on ice.
4. Centrifuge the sample at 10,000 × g for 5 minutes to pellet cell/tissue debris.
5. Transfer supernatant to a new microcentrifuge tube and discard the pellet.

Determination of protein concentration using BCA Protein Assay

6. Prepared diluted albumin (BSA) standards. Alternatively: Use pre-diluted BSA standards.
7. Prepare Working Reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1 Reagent A:B).
8. Pipette 25 µL of each standard or unknown sample replicate into a microplate well (working range = 20–2,000 µg/mL).
9. Add 200 µL of the Working Reagent to each well and mix plate thoroughly on a plate shaker for 30 seconds.
10. Cover plate and incubate at 37°C for 30 minutes.
11. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader.
12. Determine protein concentration of each cell lysate. Determine how much protein to load (recommended <50 µg/well).

Protocol tip

Preparing samples for protein electrophoresis

Denaturing electrophoresis (SDS-PAGE)

1. Prepare your sample in the appropriate sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
SDS/LDS sample buffer (4X)*	2.5 μL	2.5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

*For tricine gels, it is recommended to use a Tricine SDS sample buffer.

2. Heat samples at 70˚C for 10 minutes.
3. Load samples onto a Bis-Tris, Tris-acetate, or Tris-glycine protein gel.

Non-denaturing electrophoresis (Native-PAGE)

1. Prepare your sample in the native sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
Native sample buffer (2X)	5 μL	5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

2. Load samples onto a Tris-glycine or Tris-acetate protein gel.

Protocol tip

Do not store reduced samples. Prepare sample stock (as in the example table except for Reducing Agent) and store it at -20ºC or -80ºC. Reduce the sample with Reducing Agent (10X) prior to use.

Cell culture sample preparation

Preparation of lysate from cell culture for western blot analysis

Materials

Cell lysis buffer (see table below for recommendations)	Sample buffer (loading buffer) (e.g., LDS Sample Buffer, 4x, Cat. No. NP0007)
Protease and phosphatase inhibitor cocktail (e.g., Halt Protease and Phosphatase Inhibitor Cocktail, Cat. No. 78440 or Pierce Protease and Phosphatase Inhibitor Tablet, Cat. No. A32961)	Optional: Sample reducing agent (e.g., NuPAGE Sample Reducing Agent (10X), Cat. No. NP0004)
Cold phosphate-buffer saline (PBS) (e.g., Phosphate-Buffered Saline Buffer Packs, Cat. No. 28372)	Performing native PAGE: Native sample buffer (e.g., Tris-Glycine Native Sample Buffer, Cat. No. LC2673)
Protein assay (e.g., Pierce BCA Protein Assay, Cat. No. 23225)

View or download buffer recipes

Lysis buffer recommendations

Target protein location	Buffer recommended	Description
Whole cell (total protein extraction) (mild lysis buffer to retain protein-protein interactions)	M-PER Mammalian Protein Extraction Reagent	Non-denaturing detergent in 25mM bicine buffer (pH 7.6)
Whole cell (total protein extraction) (protein is membrane bound, nuclear or mitochondria)	RIPA Lysis Buffer	25 mM Tris-HCl pH 7.6 150 mM NaCl 1% NP-40 or Triton X-100 1% sodium deoxycholate 0.1% SDS
Cytoplasmic	NP-40 Cell Lysis Buffer	50 mM Tris, pH 7.4 250 mM NaCl 5 mM EDTA 50 mM NaF 1 mM Na₃VO₄ 1% NP-40

If working with non-mammalian cells, explore the recommended sample type-specific lysis buffers.

Procedure

1. Prepare lysis buffer by adding protease and phosphatase inhibitors. If using Halt Protease and Phosphatase Inhibitor Cocktail 100x, add 10 µL/mL of cocktail directly to the cell lysis buffer. If using Pierce Protease and Phosphatase Inhibitor tablet, dispense tablet from vial and place into 10 mL of lysis buffer and vortex to dissolve. Optional: To inhibit metalloproteases, add EDTA (0.5 M) at 10 µL/mL of lysis buffer.

For adherent cells

2. Place the cell culture dish on ice and carefully remove culture medium and wash the cells with ice-cold PBS.
3. Aspirate PBS and add ice-cold lysis buffer (~1 mL per 10⁷ cells or 100 mm plate; ~0.5 mL for 60 mm plate; ~200-400 µL for 6-well culture plate). Gently shake or swirl for 5 minutes on ice. Alternatively: Cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer in a microcentrifuge tube.
4. Collect the lysate and transfer to a microcentrifuge tube. Centrifuge samples at ~14,000 x g for 15 minutes to pellet the cell debris.
5. Transfer supernatant to a new microcentrifuge tube and discard the pellet.

For suspension-cultured cells

2. Pellet the suspension of cells by centrifugation at 2,500 x g for 10 minutes. Discard the supernatant.
3. Wash the cells once by resuspending the cell pellet in ice-cold PBS. Pellet cells by centrifugation at 2,500 x g for 10 minutes.
4. Add ice-cold lysis buffer (~1 mL per 100 mg or ~100 µL of wet cell pellet). Pipette the mixture up and down to resuspend the pellet.
5. Shake mixture gently for 10 minutes. Remove cell debris by centrifugation at ~14,000 x g for 15 minutes.
6. Transfer supernatant to a new microcentrifuge tube and discard the pellet.

Determination of protein concentration using BCA Protein Assay

7. Prepared diluted albumin (BSA) standards. Alternatively: Use pre-diluted BSA standards.
8. Prepare Working Reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1 Reagent A:B).
9. Pipette 25 µL of each standard or unknown sample replicate into a microplate well (working range = 20–2000 µg/mL).
10. Add 200 µL of the Working Reagent to each well and mix plate thoroughly on a plate shaker for 30 seconds.
11. Cover plate and incubate at 37°C for 30 minutes.
12. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader.
13. Determine protein concentration.

Protocol tip

Preparing samples for protein electrophoresis

Denaturing electrophoresis (SDS-PAGE)

Prepare your sample in the appropriate sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
SDS/LDS sample buffer (4X)*	2.5 μL	2.5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

*For tricine gels, it is recommended to use a Tricine SDS sample buffer.

2. Heat samples at 70˚C for 10 minutes.
3. Load samples onto a Bis-Tris, Tris-acetate, or Tris-glycine protein gel.

Protocol tip

Non-denaturing electrophoresis (Native-PAGE)

Prepare your sample in the appropriate sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
Native sample buffer (2X)	5 μL	5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

2. Load samples onto Tris-glycine or Tris-acetate protein gel.

Tissue sample preparation

Preparation of lysate from tissues for western blot analysis

Materials

Cell lysis buffer (see table below for recommendations)	Sample buffer (loading buffer) (e.g., LDS Sample Buffer, 4x, Cat. No. NP0007)
Protease and phosphatase inhibitor cocktail (e.g., Halt Protease and Phosphatase Inhibitor Cocktail, Cat. No. 78440 or Pierce Protease and Phosphatase Inhibitor Tablet, Cat. No. A32961)	Optional: Sample reducing agent (e.g., NuPAGE Sample Reducing Agent (10X), Cat. No. NP0004)
Cold phosphate-buffer saline (PBS) (e.g., Phosphate-Buffered Saline Buffer Packs, Cat. No. 28372)	Performing native PAGE: Native sample buffer (e.g., Tris-Glycine Native Sample Buffer, Cat. No. LC2673)
Protein assay (e.g., Pierce BCA Protein Assay, Cat. No. 23225)

View or download buffer recipes

Lysis buffer recommendations

Target protein location	Buffer recommended	Description
Whole cell (total protein extraction)	T-PER Tissue Protein Extraction Reagent (mild lysis buffer to retain protein-protein interactions and minimize denaturation)	Non-denaturing detergent in 25mM bicine, 150mM sodium chloride (pH 7.6)
Whole cell (total protein extraction) (protein is membrane-bound, nuclear, or mitochondrial)	RIPA Lysis Buffer (contains iconic detergents to assist with bringing proteins into solution)	25 mM Tris-HCl pH 7.6 150 mM NaCl 1% NP-40 or Triton X-100 1% sodium deoxycholate 0.1% SDS

Procedure

1. Prepare lysis buffer by adding protease and phosphatase inhibitors. If using Halt Protease and Phosphatase Inhibitor Cocktail 100x, add 10 µL/mL of cocktail directly to the cell lysis buffer. If using Pierce Protease and Phosphatase Inhibitor tablet, dispense tablet from vial and place into 10 mL of lysis buffer and vortex to dissolve. Optional: To inhibit metalloproteases, add EDTA (0.5 M) at 10 µL/mL of lysis buffer.
2. Dissect the tissue of interest on ice and weigh samples. Use a ratio of ~50 mg tissue to 1,000 µL of ice-cold lysis buffer. Use a smaller volume of lysis buffer if a more concentrated protein extract is required.
3. Add the appropriate amount of ice-cold lysis buffer to the tissue sample and homogenize on ice.
4. Centrifuge the sample at 10,000 × g for 5 minutes to pellet cell/tissue debris.
5. Transfer supernatant to a new microcentrifuge tube and discard the pellet.

Determination of protein concentration using BCA Protein Assay

6. Prepared diluted albumin (BSA) standards. Alternatively: Use pre-diluted BSA standards.
7. Prepare Working Reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1 Reagent A:B).
8. Pipette 25 µL of each standard or unknown sample replicate into a microplate well (working range = 20–2,000 µg/mL).
9. Add 200 µL of the Working Reagent to each well and mix plate thoroughly on a plate shaker for 30 seconds.
10. Cover plate and incubate at 37°C for 30 minutes.
11. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader.
12. Determine protein concentration of each cell lysate. Determine how much protein to load (recommended <50 µg/well).

Protocol tip

Preparing samples for protein electrophoresis

Denaturing electrophoresis (SDS-PAGE)

1. Prepare your sample in the appropriate sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
SDS/LDS sample buffer (4X)*	2.5 μL	2.5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

*For tricine gels, it is recommended to use a Tricine SDS sample buffer.

2. Heat samples at 70˚C for 10 minutes.
3. Load samples onto a Bis-Tris, Tris-acetate, or Tris-glycine protein gel.

Non-denaturing electrophoresis (Native-PAGE)

1. Prepare your sample in the native sample buffer, such that the final concentration of the sample buffer is 1X. Scale the below volumes based on the well loading volume.

Reagent	Reduced sample	Non-reduced sample
Protein sample	x μL	x μL
Native sample buffer (2X)	5 μL	5 μL
Reducing agent (10X)	1 μL	—
Deionized water	to 10 μL	to 10 μL
Total volume	10 μL*	10 μL*

2. Load samples onto a Tris-glycine or Tris-acetate protein gel.

Protocol tip

Do not store reduced samples. Prepare sample stock (as in the example table except for Reducing Agent) and store it at -20ºC or -80ºC. Reduce the sample with Reducing Agent (10X) prior to use.

For Research Use Only. Not for use in diagnostic procedures.