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Choose western blot buffers based on application needs and performance requirements such as sensitivity and reproducibility. Compare transfer, blocking, wash, and stripping buffers to reduce background and improve signal clarity.
Explore the western blot workflow
Overview of western blot buffers
Western blotting success depends on more than antibodies and detection reagents—the buffers used at each step of the workflow directly influence sensitivity, background, and reproducibility. From efficient protein transfer to clean antibody detection and membrane reprobing, selecting the right buffer is important for generating reliable, publication-quality results.
Western blot buffer types
Western blotting workflows typically use multiple types of buffers, each designed to support a specific step in the process, and can have an impact on transfer efficiency, background noise, and signal quality.
- Transfer buffers—support efficient protein transfer from gel to membrane
- Blocking buffers—minimize nonspecific binding before antibody incubation
- Wash buffers—remove unbound antibody prior to detection
- Stripping buffers—strip/remove antibodies from membrane for membrane reprobing
Each buffer type contributes to overall assay performance by influencing protein stability, antibody specificity, detection sensitivity, and reproducibility. Selecting the appropriate buffer for each step from the beginning results in less downstream optimization and improves confidence in experimental results.
The table below summarizes when to use each buffer type, typical formulations, and commonly used options within the western blotting workflow.
| Step | Transfer buffers | Blocking buffers | Wash buffers | Stripping buffers |
|---|---|---|---|---|
| When to use | Gel-to-membrane transfer | Reduce background before antibody binding | Remove unbound antibody | Reprobing membranes |
| Buffer type | Tris-glycine (Towbin), Bis-Tris, Tris-acetate | Milk, BSA, casein, specialized blocker | TBS, TBS-T, PBS, PBS-T | Mild or harsh stripping buffer |
| Common composition | With or without methanol/SDS | 3–5% in TBS-T/PBS-T | 0.05–0.1% Tween-20 | Various |
| Related products |
Need a running buffer? Protein electrophoresis buffers and reagents.
Western blot transfer buffers guide
Quick selection guide: Which transfer buffer should you use?
- Using Bis-Tris gels (Bolt or NuPAGE)? Choose Bolt or NuPAGE Bis-Tris transfer buffer
- Working with small proteins (<40 kDa)? Choose Novex Tris-Glycine transfer buffer
- Working with large proteins (>150 kDa)? Choose NuPAGE Bis-Tris transfer buffer
- Need fast transfer? Choose semi-dry 1-step transfer buffers
How to choose the right transfer buffer
Selecting the right transfer buffer depends on your gel chemistry, protein size, and transfer method.
- Match your gel chemistry (Tris-Glycine, Tricine, Bis-Tris, or Tris-Acetate)
- Select transfer method (wet = flexibility, semi-dry = speed)
- Optimize conditions (standard vs. high ionic strength buffers)
Compare western blot transfer buffers
The table below summarizes recommended transfer buffers based on gel chemistry, protein size, and transfer method.
| Wet transfer | Semi-dry transfer | ||||||
| Transfer buffer | Novex Tris-Glycine transfer buffer | Bolt Bis-Tris transfer buffer Reduced samples: Antioxidant* |
NuPAGE Bis-Tris transfer buffer Reduced samples: Antioxidant* |
Power Blotter 1-Step Transfer Buffer (5X)** | Pierce 1-Step Transfer Buffer (1X)** | ||
| When to use | General-purpose wet transfer for broad protein size ranges | Controlled transfer of low-molecular-weight proteins resolved on Tricine gels | Optimized transfer performance for Bolt Bis-Tris Plus gel systems | Consistent transfer optimized for NuPAGE Bis-Tris gel chemistry | Efficient transfer of high-molecular-weight proteins from Tris-Acetate gels | High-ionic-strength buffer enabling rapid semi-dry protein transfer | Ready-to-use buffer for fast, reproducible semi-dry transfers |
| Gel chemistry | Novex Tris-Glycine | Novex Tricine | Bolt Bis-Tris Plus | NuPAGE Bis-Tris | NuPAGE Tris-Acetate | All gel chemistries | |
*Antioxidant prevents re-oxidation of reduced proteins during transfer, improving band integrity
**1-Step Transfer buffer is a high ionic strength formulation which allows for 5-minute to 12-minute protein transfer when used with compatible semi-dry blotting systems. 1-Step Transfer Buffer is compatible with Power Blotter and other protein semi-dry transfer devices, when they are paired with a suitable high-current power supply. Such devices should help provide constant high current (1.3 to 5.0 amps) to rapidly transfer proteins via the high ionic strength conditions supplied by the transfer buffer.
What do western blot transfer buffers do?
Protein transfer is a critical step in western blotting. Transfer buffers help provide the ionic strength and pH required to move proteins efficiently from the gel to a membrane while preserving protein integrity and epitope accessibility. The buffer composition directly affects transfer efficiency, speed, and uniformity, particularly across different molecular weight ranges. Transfer buffers also affect compatibility with nitrocellulose or PVDF membranes and determine whether proteins remain bound during downstream detection steps.
Why transfer buffers matter
Using a premixed transfer buffer matched to your gel chemistry and workflow helps to:
- Support efficient electrotransfer under wet and semi-dry conditions
- Maintain protein solubility and structural integrity
- Improve consistency across low-, broad-, and high-molecular-weight proteins
- Reduce incomplete transfer or protein loss
When to choose ready-to-use vs. DIY transfer buffers
Thermo Fisher Scientific offers premixed, concentrated, and specialized transfer buffers optimized for different gel chemistries and transfer systems, helping ensure consistent results while reducing preparation time.
- Choose ready-to-use transfer buffers when consistency, speed, and reproducibility are critical, especially in high-throughput or multi-user labs.
- Prepare transfer buffers from scratch when protocols require customization or cost control is a priority.
Tip: For most workflows, matching a ready-to-use transfer buffer to your gel system helps provide the fastest path to reliable results.
The following transfer buffer recipes are provided to allow preparation of buffers from scratch.
25X Tris-Glycine transfer buffer
1. Dissolve the following reagents in 400 mL ultrapure water.
| Reagent | Amount | Concentration (1X) |
|---|---|---|
| Tris Base | 18.2 g | 12 mM |
| Glycine | 90 g | 96 mM |
2. Mix well and adjust volume to 500 mL with ultrapure water.
3. Before western transfer, dilute to 1X with water.
4. Buffer is stable for 6 months at room temperature.
20X Bis-Tris transfer buffer
1. Dissolve the following reagents in 100 mL ultrapure water.
| Reagent | Amount | Concentration (1X) |
|---|---|---|
| Bicine | 10.2 g | 25 mM |
| Bis-Tris (free base) | 13.1 g | 25 mM |
| EDTA | 0.75 g | 1 mM |
2. Mix well and adjust volume to 125 mL with ultrapure water.
3. Before western transfer, dilute to 1X with water.
4. Buffer is stable for 6 months when stored at 4°C.
Western blot blocking buffers guide
Quick selection guide: Which blocking buffer should you use?
- General chemiluminescent western blotting? Choose StartingBlock Blocking Buffer.
- High background or low signal with milk? Switch to Blocker Casein.
- Detecting phosphoproteins or using biotin/streptavidin systems? Choose Blocker BSA.
- Fluorescent western blotting or imaging? Choose Blocker FL Fluorescent Blocking Buffer.
How to choose the right blocking buffer
Selecting the right blocking buffer depends on your detection method, nature of the target protein, background levels, and signal intensity.
- Choose your detection method (chemiluminescent vs fluorescent western blotting).
- Consider your target protein (phosphoproteins, biotin/streptavidin systems, or routine targets).
- Evaluate background and sensitivity (high background = switch blockers; weak signal = adjust blocking conditions).
Compare western blot blocking buffers
The table below summarizes recommended blocking buffers based on detection method, target type, and experimental conditions.
|
Chemiluminescent western blotting (HRP/AP) |
Fluorescent western blotting |
||
Blocking buffer |
||||
Why use |
General-purpose blocker for chemiluminescent detection |
Improved sensitivity, high-performance replacement for milk-based blockers |
Phosphoproteins or biotin-based chemiluminescent detection |
Performing fluorescent western detection |
Blocking agent |
Serum- and biotin-free single purified protein |
Purified protein |
Purified bovine serum albumin |
Single purified protein |
Highlights |
|
Single protein blocking buffer helps provide fewer chances of cross-reaction with assay components than serum or milk solutions |
|
|
Available formats |
PBS, TBS, PBST, TBST |
PBS, TBS |
PBS, TBS |
10X concentrate |
User guides |
||||
Tip: If changing antibody concentration doesn’t improve results, change the blocking buffer before changing the antibody.
What do western blot blocking buffers do?
After protein transfer, membranes contain both immobilized target proteins and unoccupied binding sites. If these sites are not blocked, primary or secondary antibodies can bind nonspecifically, leading to elevated background and reduced signal-to-noise ratio. Blocking buffers coat these remaining unoccupied binding sites so antibodies bind selectively to their intended targets, preventing nonspecific binding, reducing background and improving signal clarity.
An effective blocking buffer binds nonspecific interaction sites without masking the target epitope or interfering with antibody binding.
Which Blocking Buffer Should I Use for Each Detection Method?
| Detection method | Recommended Thermo Fisher Blocking Buffers | Why |
|---|---|---|
| Chemiluminescent (HRP) | StartingBlock Blocking Buffer |
Compatible with HRP detection and reduces nonspecific binding |
| Alkaline phosphatase (AP) | SuperBlock Blocking Buffer | Phosphate inhibits AP; use TBS-based formulations |
| Fluorescent (NIR) | Blocker FL Fluorescent Blocking Buffer | Minimizes autofluorescence and imaging background |
| Multiplex fluorescence | Blocker FL Fluorescent Blocking Buffer | Supports low-background detection of multiple targets |
| Biotin/streptavidin-based detection | StartingBlock Blocking Buffer; SuperBlock Blocking Buffer; Protein-Free Blocking Buffer | Avoids endogenous biotin interference |
Blocking buffer formats and options
Thermo Fisher provides blocking buffer in multiple formats to support different workflows:
- Ready-to-use blocking buffers for convenience and consistency
- Concentrated formats for flexible dilution
- Blocking buffers optimized for chemiluminescent and fluorescent detection
- Single-protein blockers designed to reduce cross-reactivity or improve sensitivity
These formats support applications such as:
- Low-background imaging
- Multiplex fluorescence detection
- High-sensitivity chemiluminescence
Blocking buffer timing and conditions
Typical blocking times range from 15 to 60 minutes at room temperature, depending on the blocking buffer used. Increasing blocking time does not always reduce background and may reduce sensitivity.
5% nonfat milk (for routine chemiluminescent detection)
| Nonfat dry milk | 2.5 g |
| 1X TBST or 1X PBST | Up to 50 mL |
| Filter to remove particulates |
Ready-to-use alternative:
3% BSA (phosphoproteins or biotin-based assays)
| BSA | 1.5 g |
| 1X TBST or 1X PBST | Up to 50 mL |
| Filter to remove particulates |
Ready-to-use Thermo Fisher blocking buffers are recommened when reproducibility, convenience, or reduced optimization time is critical.
Ready-to-use alternatives: Pierce Blocker BSA (10X) in TBS and Pierce Blocker BSA (10X) in PBS.
Western blot wash buffers guide
Quick selection guide: Which wash buffer should you use?
- Standard HRP-based western blot washing? Use PBS or PBST (PBS + Tween-20).
- Need to reduce background (HRP or AP workflows)? Use TBST (TBS + Tween-20).
- Alkaline phosphatase (AP) detection? Use TBS (avoid PBS, which inhibits AP).
- High background? Use TBST or PBST with 0.05–0.1% Tween-20.
- Persistent background? Increase Tween-20 concentration (up to ~0.2%).
- Fluorescent western blotting? Use detergent-free buffers (TBS or PBS).
How to choose the right wash buffer
Choosing the right wash buffer depends on your detection chemistry, need for detergent, and background vs. signal balance.
- Match your detection chemistry (HRP → PBS or TBS; AP → TBS only).
- Decide if detergent is needed (high background → add Tween-20; weak signal → reduce or omit detergent).
- Optimize detergent concentration (Typical: 0.05–0.1% Tween-20; increase for high background, decrease for weak signal).
- Consider imaging requirements (fluorescent detection → avoid detergents during blocking; use only after blocking step, as needed).
Compare western blot wash buffers
The table below summarizes common wash buffers and formats based on detection method, background control, and workflow requirements.
Choose from dry blend packs or concentrated solutions (10X or 20X) of common wash buffers used in western blotting.
|
TBS |
PBS |
TBST (+ Tween-20) |
PBST (+ Tween-20) |
Best for |
Alkaline phosphatase (AP) –based detection and detergent-free washing |
General HRP-based chemiluminescent western blot washing |
Reducing nonspecific background in HRP or AP detection workflows |
Routine HRP detection for removing potential high background/unbound reagents |
Dry blend |
|
|
||
Liquid conc. |
||||
Formulation |
|
|
|
|
For use with fluorescent systems |
Yes |
Yes |
No |
|
Tip: If background remains high, increase wash number or duration before increasing detergent concentration.
What do western blot wash buffers do?
Wash buffers remove unbound antibodies and reagents between incubation steps, reducing background while preserving specific signal. Insufficient washing produces high background, while excessive washing may result in decreased sensitivity caused by stripping of the antibody and/or antigen from the blot.
Washing is performed in physiological buffers such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS). Detergents such as Tween-20 can be added to the buffer to help remove nonspecifically bound material. The amount of Tween-20 (0.05%–0.2%) will vary depending on the strength of the antibodies used. Weak binding antibodies may be washed away by too much detergent.
Why wash buffers matter
Effective washing helps to:
- Reduce nonspecific signal by removing loosely or nonspecifically bound antibodies that contribute to background
- Improve assay sensitivity by increasing the signal-to-noise ratio, making true target bands easier to detect
- Maintain consistent signal across replicates by ensuring uniform removal of excess reagents
Insufficient washing can result in high background and false-positive signals, while overly stringent washing may reduce sensitivity by removing weakly bound antibodies or target proteins.
Wash protocol recommendations
- Typical washes: 3–5 washes, 5–10 minutes each
- Increase detergent or wash number for high background
- Reduce detergent concentration if signal is weak
Tris-buffered saline with Tween 20 (TBST)
| 10X TBS | 100 mL |
| Tween 20 | 1 mL |
| Deionized water | to 1,000 mL |
Phosphate buffered saline with Tween 20 (PBST)
| 10X TBS | 100 mL |
| Tween 20 | 1 mL |
| Deionized water | to 1,000 mL |
Western blot stripping buffers guide
Quick selection guide: Which stripping buffer should you use?
- Need gentle stripping for sensitive or low-affinity antibodies? Use Restore Western Blot Stripping Buffer.
- Removing high-affinity antibodies or performing multiple reprobes? Use Restore PLUS Western Blot Stripping Buffer.
- Fluorescent (NIR) western blot detection? Use Restore Fluorescent Western Blot Stripping Buffer.
- Working with valuable or limited samples? Choose Restore Stripping Buffer to preserve immobilized protein.
- Need faster or more complete antibody removal? Use Restore PLUS Stripping Buffer or extended protocol.
How to choose the right stripping buffer
Choosing the right stripping buffer depends on antibody affinity, detection method, and how well the membrane must be preserved.
- Determine if stripping is necessary (multiple targets, antibody optimization, or limited samples).
- Assess antibody affinity (low/moderate → mild buffer; high affinity → stronger buffer).
- Match your detection method (chemiluminescent → standard buffers; fluorescent → fluorescence-compatible buffers).
- Protect membrane integrity (minimize time, temperature, and number of stripping cycles).
Compare western blot stripping buffers
The table below summarizes stripping buffers based on antibody affinity, detection method, and membrane compatibility.
| Restore Stripping Buffer | Restore PLUS Stripping Buffer | Restore Fluorescent Western Blot Stripping Buffer | |
|---|---|---|---|
| Type of buffer | Standard/mild | Strong | Fluorescent-compatible |
| When to use | For routine and sensitive/low-affinity antibodies | For removing high-affinity primary antibodies | For removing fluorescent antibodies |
| Features | Gentle, odor-free | • Robust yet gentle, odor free •Transferred proteins remain viable and can strip the same blot up to 5 times |
• Gentle and highly effective for removing primary and fluorescent secondary antibodies • Optimized for fluorescent western blot workflows |
| Membrane | NC and PVDF | NC and PVDF | Low-fluorescence PVDF membranes recommended |
| Time of incubation | 15–30 min at 37°C | 5–15 mins at RT or 37°C for high affinity antibodies | 10–20 min at RT |
| User guide | Restore Western Blot Stripping Buffer User Guide | Restore Plus Western Blot Stripping Buffer User Guide | Restore FL Western Blot Stripping Buffer User Guide |
Technical tip: Strip and reprobe western blots
Follow step-by-step guidance for stripping antibodies and reprobing western blot membranes.
What do western blot stripping buffers do?
Stripping buffers are designed to remove bound primary and secondary antibodies from western blots so that membranes can be reprobed under alternate conditions or with another antibody to detect a different protein target, without rerunning another western blot. Reprobing a western blot provides an efficient way to analyze additional targets while conserving samples and allowing optimization as needed.
Types of stripping buffers
Stripping buffers vary in strength and formulation to support different antibodies, detection methods, and membrane reuse needs. Choosing the appropriate type helps ensure effective antibody removal while preserving immobilized proteins.
- Mild stripping buffers: For low- to moderate-affinity antibodies or sensitive targets, helping provide gentle antibody removal with minimal protein loss
- Recommended for limited reprobing cycles
- Strong stripping buffers: For high-affinity or difficult-to-remove antibodies, offering more aggressive stripping when complete antibody removal is required
- Recommended when complete antibody removal is required and target protein abundance is sufficient
- Fluorescent-compatible stripping buffers: Optimized for fluorescent western blotting, including near-infrared (NIR) detection, enabling efficient removal of dye-labeled antibodies while minimizing membrane autofluorescence
Tip: Start with the mildest stripping buffer that effectively removes antibodies and increase strength only if needed.
When to strip and reprobe
Stripping is recommended when:
- Antibody optimization is required
- Sample availability is limited
Advantages of stripping and reprobing blots
There are several major reasons to choose to strip and reprobe a western blot. Following are some of the most important:
- Conserves sample: When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed with the same or different antibodies.
- Improves workflow efficiency: Reprobing allows for the detection of multiple proteins from the same blot, reducing the need to repeat earlier western blot workflow steps.
- Facilitates assay optimization: The light emission intensity of high-sensitivity chemiluminescent substrates often require antibody concentration optimization to achieve the highest quality blot. Optimization is achieved easily by stripping the membrane and reprobing with different antibody concentration.
- Allows for correcting mistakes: Immunoblotting requires many steps, giving ample opportunity for mistakes to occur. By stripping the membrane, the blot can be reused.
| 0.5 M Tris HCl, pH 6.8 | 12.5 mL |
| 10% SDS | 20 mL |
| 2-mercaptoethanol | 0.8 mL |
| Deionized water | 67.5 mL |
How-to videos
How do I probe a western blot?
Probing is a critical stage of western blotting that determines signal specificity, sensitivity, and background. In this video, you’ll learn how to correctly probe a western blot after protein transfer, including blocking, antibody incubation, and washing steps, to help ensure reliable and reproducible detection.
You’ll learn how to:
- Select and apply the appropriate blocking buffer to reduce nonspecific binding
- Prepare and dilute primary and secondary antibodies
- Perform effective washing steps to minimize background and preserve signal
How do I strip and reprobe my western blot?
Stripping and reprobing allows a single western blot membrane to be reused for detection of multiple targets, helping save sample and reagents. In this video, you’ll learn how to safely remove bound antibodies and reprobe the membrane without compromising immobilized proteins or signal quality.
You’ll learn how to:
- Select the appropriate stripping buffer based on antibody affinity and detection method
- Optimize stripping conditions to remove antibodies while preserving target proteins
- Handle membranes during stripping to preserve immobilized proteins
Ordering information
Frequently asked questions
Blocking buffer selection depends on detection method and target protein. Milk-based blockers work well for routine chemiluminescent detection. BSA or purified-protein blockers are preferred for phosphoproteins or biotin-based assays. Fluorescent western blotting requires fluorescence-compatible blocking buffers to minimize background.
High-molecular-weight proteins transfer more efficiently when using gel chemistries and transfer buffers designed for large proteins, such as Tris-Acetate systems. For wet transfer, reduce methanol to 10–15% and add up to 0.02% SDS to improve mobility. Additionally, increased transfer time and using PVDF membranes can enhance binding and overall transfer efficiency.
PBS-T and TBS-T are suitable for most western blots. However, TBS-T is recommended for alkaline phosphatase (AP) detection and phosphorylation studies because phosphate in PBS can interfere with AP-based systems. Choose the buffer based on detection chemistry and background performance.
Most western blot workflows use 0.05–0.1% Tween-20 in TBS or PBS. If background is high increase detergent up to 0.2% if signal is weak reduce detergent 0.02–0.05%. Alternatively, adjust wash duration or frequency before significantly increasing detergent concentration.
Strip and reprobe a western blot when analyzing multiple targets, optimizing antibodies, or conserving limited samples. Use mild stripping buffers for low-affinity antibodies and stronger buffers for high-affinity antibodies. PVDF membranes are generally preferred for repeated stripping due to their durability.
Reusing transfer buffer is not recommended. Ion depletion, pH changes, methanol loss, and heat generation alter buffer performance during transfer. Using fresh buffer helps ensure consistent transfer efficiency and reproducible results.
Consider using an antioxidant when you need enhanced sensitivity or improved band quality, particularly for low-abundance or difficult-to-transfer proteins or challenging targets. Antioxidants help improve protein transfer efficiency, which can result in stronger signal intensity, better sensitivity, and sharper, more defined bands.
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Western blot educational resources
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