Western blotting (WB), also known as protein immunoblotting, is one of the most widely used techniques for protein analysis in life science research. The core workflow involves separating protein samples by polyacrylamide gel electrophoresis (PAGE), transferring proteins onto a solid membrane, and detecting target proteins using specific antibodies (typically a primary antibody followed by a labeled secondary antibody).

Due to its high sensitivity and strong specificity, Western blotting has become a fundamental tool for studying protein expression, signaling pathways, and post-translational modifications. Among all WB steps, protein sample preparation is the very first and one of the most critical steps, as its quality directly determines the reliability, reproducibility, and interpretability of downstream results.

Why Protein Extraction Matters in Western Blotting

The primary goal of protein extraction is to maximize recovery of intact target proteins while minimizing degradation or modification during sample preparation. To achieve this, protein extraction should generally be performed:

• At low temperatures
• In short time frames
• Using appropriate lysis buffers and inhibitors

Because different samples (e.g., tissues vs. cultured cells) have distinct biochemical properties, no single lysis strategy fits all experiments. Selecting the right extraction method and buffer is therefore essential.

Know Your Target Protein Before You Start

Before protein extraction, researchers should gather as much information as possible about the target protein, including:

• Expression level (high vs. low abundance)
• Subcellular localization (cytoplasmic, nuclear, membrane-bound, etc.)
• Molecular weight range
• Presence of alternative splicing or post-translational modifications

This information helps guide:

• Selection of lysis buffer
• Choice of loading amount
• Optimization of electrophoresis and transfer conditions
• Selection of appropriate loading controls

A well-informed extraction strategy significantly increases the chance of obtaining clean, interpretable WB results.

Commonly Used Lysis Buffers for Western Blotting

Selecting the appropriate lysis buffer is a critical step in protein extraction. Typical lysis buffers contain:

A buffering system, Salts, Detergents, Protease and/or phosphatase inhibitors. Each component plays a specific role and collectively determines extraction efficiency and protein integrity.

Lysis Buffer	Key Features	Typical Applications
RIPA Buffer	Strong lysis capability; contains ionic and non-ionic detergents	Total protein extraction; nuclear and membrane proteins
NP-40 / Triton X-100 Buffer	Mild, non-ionic detergents	Cytoplasmic proteins; protein–protein interaction studies
SDS Lysis Buffer	Very strong denaturing conditions	Insoluble or aggregated proteins
Nuclear/Cytoplasmic Extraction Buffers	Fractionation of cellular compartments	Subcellular localization studies

[Note]:

For special downstream applications (e.g., mass spectrometry), certain inhibitors such as AEBSF should be avoided, as they may cause mass spectral peak shifts. For nitric oxide (NO)–related assays, protease and phosphatase inhibitors may also interfere with results. In such cases, lysis buffers without pre-added inhibitors (e.g., Cat. No. 20119ES or 20120ES) are recommended, allowing users to customize inhibitor composition.

Understanding RIPA Buffer Components

RIPA buffer is one of the most commonly used lysis buffers for Western blotting. Its typical components include:

• Tris-HCl (pH ~7.4): Provides a stable buffering environment close to physiological pH.
• NaCl (physiological concentration): Maintains protein solubility and preserves protein–protein interactions.
• EDTA: Chelates metal ions and inhibits metalloproteases.
• SDS: A strong anionic detergent that disrupts membranes and denatures proteins. Higher salt concentrations can enhance SDS denaturation effects.
• Sodium deoxycholate: A milder anionic detergent that aids membrane disruption.
• Triton X-100: A non-ionic detergent that solubilizes membranes without denaturing proteins or disrupting protein–protein interactions.

Understanding these components helps researchers fine-tune lysis conditions based on experimental needs.

Key Precautions During Protein Extraction

1. Prevent Protein Degradation

For mammalian tissues or cells, perform the entire extraction workflow at 4°C or on ice to suppress protease activity. Work efficiently and minimize processing time.

Tissue Collection Order and Handling

To reduce cross-contamination and degradation:

• Process digestive enzyme–rich tissues (e.g., liver, pancreas, stomach, intestine) first
• Follow with immune cell–rich tissues (e.g., lung)
• Collect reproductive tissues next
• Process heart, spleen, kidney, and brain last

If tissues cannot be processed immediately, snap-freeze in liquid nitrogen or store at −80°C. Fresh tissue is strongly preferred, especially for digestive organs, as it typically yields stronger WB signals with lower background. Samples subjected to repeated freeze–thaw cycles should be discarded.

2. Reducing Interference from Contaminants

Protein lysates commonly contain contaminants that may compromise the resolution and quality of subsequent electrophoresis. The following strategies can be employed to improve this step:

Type of Contaminant	Recommended Solution
Exogenous proteins	Ensure all labware and reagents are clean and free of protein contamination. For adherent cells, avoid trypsin digestion whenever possible; instead, lyse cells directly or collect by scraping.
Nucleic acids (increase viscosity and hinder sample loading)	Fragment genomic DNA by brief sonication or repeated shearing using a 1 mL syringe and needle. Alternatively, add a broad-spectrum nuclease (e.g., Universal Nuclease, Cat. No. 20156ES) to digest nucleic acids.
Lipids	Carefully aspirate the aqueous protein phase while avoiding the lipid layer. If necessary, remove the lipid layer prior to protein collection.
Excess salts	High salt concentrations can cause “smiling” bands and uneven migration. Keep salt concentrations within an appropriate range and ensure consistent ionic strength across all samples.

3. Effects of Trypsin on Membrane Proteins

When studying membrane proteins, be aware that trypsin used during cell passaging can cleave extracellular domains, leading to: Signal loss or Unexpected band sizes

Recommendations:

• Avoid trypsinization during the final passage
• Allow cells sufficient recovery time for membrane protein re-expression
• Collect adherent cells by direct lysis or cell scraping rather than enzymatic detachment

4. Cell or tissue disruption is a critical step in protein extraction. Common methods include mechanical homogenization, sonication, and chemical lysis. Select an appropriate method based on your sample type and ensure complete disruption.

5. Total protein lysates are suitable for detecting the expression of most cellular proteins. If isolation of organelle-specific proteins is required, use specialized subcellular fractionation kits.

6. Exercise caution when handling protease inhibitors such as PMSF, as they can be hazardous to the respiratory system, eyes, and skin. Always use appropriate personal protective equipment (PPE).

7. Protein Storage: Aliquot the extracted protein samples immediately and store them long-term at –80°C to avoid repeated freeze-thaw cycles, which can degrade protein integrity.

Successful Western blotting starts with high-quality protein extraction. By carefully selecting lysis buffers, controlling temperature and timing, and understanding the biochemical properties of both your sample and target protein, you can greatly improve the accuracy, reproducibility, and interpretability of your WB results.

Related Products

Common Lysis Buffers

Cat. No.	Product Name	Main Application	Active Lysis Components	Lysis Strength	Membrane Protein Extraction	Cytoplasmic Protein Extraction	Nuclear Protein Extraction	Protease Inhibitors Included	Phosphatase Inhibitors Included
20101ES	RIPA Lysis Buffer (Strong)	WB / IP	1% Triton X-100, 1% Sodium Deoxycholate, 0.1% SDS	Strong	Excellent	Excellent	Excellent	Yes	Yes
20114ES	RIPA Lysis Buffer (Mild)	WB / IP / Co-IP	1% NP-40, 0.25% Sodium Deoxycholate	Mild	Moderate	Excellent	Good	Yes	Yes
20115ES	RIPA Lysis Buffer (Moderate)	WB / IP	1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS	Moderate	Good	Excellent	Good	Yes	Yes

Protease Inhibitor Products

Cat. No.	Product Name	Package Size	Components	Application
20123ES	Protease Inhibitor Cocktail, EDTA-Free, Mini Tablets	1 bottle (10 tablets) / 1 bottle (50 tablets)	AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, Pepstatin A	Protein extraction from mammalian cells or tissues
20124ES	Protease Inhibitor Cocktail, EDTA-Free, 100× DMSO Stock	1 mL / 10 × 1 mL / 100 × 1 mL	AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, Pepstatin A	Protein extraction from mammalian cells or tissues