# Histone Western Blot: Protocol, Troubleshooting, and Best Practices

## Introduction to Histone Western Blotting

Histone western blotting is the immunodetection of core histone proteins (H2A, H2B, H3, H4) and their post-translational modifications (PTMs) after electrophoretic separation and transfer to a membrane. Unlike standard western blots targeting abundant cytoplasmic or nuclear proteins, histone blots present a distinct set of technical challenges rooted in the unique biochemical properties of these proteins. Histones are small (11–15 kDa), highly basic (pI > 10), and present at extraordinarily high concentrations within the nucleus—approximately 30 million copies of each core histone per mammalian cell. These properties demand specialized extraction, electrophoresis, transfer, and detection strategies.

The primary purpose of histone western blotting is to quantify specific PTMs—acetylation, methylation, phosphorylation, ubiquitination, crotonylation, and others—that collectively constitute the [histone code](/knowledge/molecular-biology/histone-code). This code regulates chromatin accessibility, transcription, DNA repair, and replication. A well-executed histone western blot allows researchers to ask whether a particular modification changes upon genetic perturbation, drug treatment, or differentiation. It also enables the detection of histone variants (e.g., H3.3, CENP-A) and the assessment of total histone levels as loading controls.

### Why Histones Require Special Western Blot Conditions

Standard Laemmli SDS-PAGE and wet transfer protocols optimized for 50–150 kDa proteins frequently fail for histones. Three properties account for this:

1. **Small molecular weight**: Histones migrate rapidly through standard percentage gels and can run off the gel if electrophoresis is prolonged. They also transfer quickly but can pass through membranes if transfer conditions are too aggressive.
2. **High basicity**: Histones are rich in lysine and arginine residues, giving them a net positive charge at physiological pH. In SDS-PAGE, SDS binding is less efficient on highly basic proteins, leading to aberrant migration and poor separation from other small basic proteins.
3. **Extensive PTM heterogeneity**: A single histone, such as H3, can carry multiple simultaneous modifications. Acetylation neutralizes lysine's positive charge, while methylation preserves it. These charge differences alter SDS binding and electrophoretic mobility, producing distinct band shifts that are diagnostically useful but also a source of confusing multiple bands if not understood.

### Applications in Epigenetics Research

Histone western blots are used to validate chromatin immunoprecipitation (ChIP) results, screen for the activity of histone-modifying enzymes such as [histone acetyltransferases](/knowledge/molecular-biology/histone-acetyltransferase) (HATs) and histone deacetylases (HDACs), assess the effects of epigenetic drugs, and confirm knockdown or overexpression of chromatin regulators. They are also essential for characterizing [histone methylation](/knowledge/molecular-biology/histone-methylation) patterns, where antibodies must distinguish between mono-, di-, and tri-methyl states on specific lysine residues. The technique is complementary to mass spectrometry: western blots provide targeted, quantitative, and relatively inexpensive validation of specific modifications, while mass spectrometry offers unbiased global profiling.

## [Histone Extraction](/knowledge/molecular-biology/histone-extraction) and Sample Preparation

The quality of a histone western blot is determined before electrophoresis begins. [Histone extraction](/knowledge/molecular-biology/histone-extraction) must yield pure, unmodified-in-vitro histones, preserve existing PTMs, and produce a sample compatible with downstream quantification.

### Acid Extraction vs. Nuclear Isolation

Two principal methods exist for isolating histones from cultured cells or tissues: acid extraction and nuclear isolation followed by salt or detergent extraction.

**Acid extraction** exploits the high basicity of histones. Cells are lysed in a hypotonic buffer containing a non-ionic detergent (e.g., 0.1% NP-40 or Triton X-100) to release nuclei. The crude nuclear pellet is then resuspended in 0.2–0.4 M HCl or 0.25 M H₂SO₄. Histones, being highly basic, dissolve readily in acid, while most other nuclear proteins precipitate. After centrifugation, the supernatant contains histones, which are then precipitated with trichloroacetic acid (TCA) at a final concentration of 20–25% (w/v) on ice for 30 minutes. The precipitate is washed with ice-cold acetone (optionally containing 0.1% HCl to remove residual TCA), air-dried, and resuspended in water or Laemmli buffer. Acid extraction yields highly purified histones but can hydrolyze certain labile modifications, particularly phosphorylation and some acetyl groups, if performed at elevated temperatures or for extended periods.

**Nuclear isolation** without acid is gentler and preserves acid-labile modifications. Cells are lysed in hypotonic buffer, nuclei are pelleted, and histones are extracted using high salt (e.g., 2 M NaCl) or mild detergents. Alternatively, micrococcal nuclease digestion can release [histone nucleosome](/knowledge/molecular-biology/histone-nucleosome) particles, from which histones are purified. This approach is preferred when studying phosphorylation (e.g., H3S10ph) or when the researcher needs to preserve the native [histone DNA](/knowledge/molecular-biology/histone-dna) interactions for downstream applications.

For most histone western blot applications, acid extraction is sufficient and is the method of choice for its speed, yield, and purity. However, if you are studying phosphorylation, consider nuclear isolation or include phosphatase inhibitors throughout.

**Critical inhibitor considerations**: Regardless of extraction method, the lysis and extraction buffers must contain:
- **Protease inhibitors**: PMSF (1 mM), aprotinin (2 µg/mL), leupeptin (2 µg/mL), and pepstatin A (1 µg/mL) to prevent histone degradation.
- **Deacetylase inhibitors**: Sodium butyrate (5–10 mM) or trichostatin A (TSA, 1 µM) to inhibit HDACs, and nicotinamide (5–10 mM) to inhibit sirtuins (SIRT1–7). These are essential when studying acetylation.
- **Phosphatase inhibitors**: Sodium orthovanadate (1 mM), sodium fluoride (10 mM), and β-glycerophosphate (10 mM) when studying phosphorylation.

### Quantification and Normalization of Histone Samples

Histone samples are notoriously difficult to quantify by standard BCA or Bradford assays because histones are basic and contain few aromatic residues, leading to underestimation by A₂₈₀ measurement and variable dye binding. The most reliable approach is to run a small aliquot on a Coomassie-stained SDS-PAGE gel alongside a known quantity of purified histone standard (commercially available from calf thymus or recombinant sources). Densitometry of the Coomassie-stained bands (particularly H3 and H4, which resolve cleanly) provides an accurate relative quantification.

Alternatively, use a detergent-compatible (DC) protein assay with a histone standard curve, or measure A₂₀₅ (peptide bond absorbance) rather than A₂₈₀. For normalization across samples, total histone H3 or total H4 levels are commonly used as loading controls. However, this is only valid if total histone levels are unchanged between conditions—an assumption that fails during replication, apoptosis, or senescence. In such cases, normalize to total protein (e.g., Ponceau S staining of the membrane) or to a housekeeping protein from the input lysate.

## Gel Electrophoresis for Histone Separation

The choice of gel system determines whether you resolve individual histone variants, separate modified from unmodified forms, and obtain sharp bands suitable for quantification.

### Gel Composition and Buffers

Standard Laemmli Tris-glycine gels (10–12%) poorly resolve histones. The small size of histones means they migrate near the dye front, and the high basicity causes smearing. Two gel systems are recommended:

**Tris-Tricine SDS-PAGE**: This system, originally developed for small proteins and peptides, uses a tricine-based trailing ion instead of glycine. The gel typically consists of a 4% stacking gel (pH 6.8) and a 16% separating gel (pH 8.45) with 6 M urea optionally added to improve resolution of modified histone forms. Tris-Tricine gels provide superior resolution of proteins below 20 kDa and are the gold standard for histone western blots. The anode buffer is 0.2 M Tris-HCl (pH 8.9), and the cathode buffer is 0.1 M Tris, 0.1 M Tricine, 0.1% SDS (pH ~8.25). Run at 100 V constant for stacking and 150 V for separation.

**Bis-Tris gels with MES or MOPS running buffer**: Commercial precast Bis-Tris gels (e.g., NuPAGE) run at neutral pH, which reduces protein modification during electrophoresis and provides sharp bands. For histones, use a 12% or 4–12% gradient gel with MES running buffer (50 mM MES, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH 7.3). MES buffer gives better separation of low-molecular-weight proteins than MOPS. Run at 200 V constant for 35–40 minutes.

For resolving histone variants (e.g., H3.1 vs. H3.3) or highly modified forms, include 6–8 M urea in the gel. Urea disrupts secondary structure and improves separation based on charge differences introduced by acetylation or phosphorylation. However, urea gels require longer polymerization times and can be brittle; handle with care.

### Running Conditions for Optimal Resolution

Load 1–10 µg of total histone protein per lane. This is far less than the 20–50 µg used for standard western blots because histones are abundant and antibodies are typically high-affinity. Overloading leads to smearing and high background. Run the gel until the dye front reaches the bottom—do not over-run, as histones will migrate off the gel. For a 16% Tris-Tricine gel, this typically takes 60–90 minutes at 150 V.

Include a pre-stained molecular weight marker with bands below 20 kDa (e.g., 10, 15, 20 kDa). Histones run anomalously: H3 (~15.3 kDa) often migrates at ~17 kDa, H2B (~13.8 kDa) at ~15 kDa, H2A (~14.0 kDa) at ~14 kDa, and H4 (~11.3 kDa) at ~10–11 kDa. The migration order on SDS-PAGE is typically H3 > H2B > H2A > H4, but this varies with gel system and acetylation status.

## Transfer and Membrane Selection

Histones are small and basic, making them prone to inefficient transfer or complete passage through the membrane. Optimization of transfer conditions is critical.

### Wet vs. Semi-Dry Transfer

**Wet (tank) transfer** is the most reliable method for histones. Use a transfer buffer of 25 mM Tris, 192 mM glycine, 10–20% methanol, pH 8.3. Methanol helps strip SDS from proteins and promotes binding to membranes, but it also causes gel shrinkage and can reduce transfer efficiency for small proteins. For histones, 10% methanol is often preferred over 20% to maintain higher transfer efficiency. Transfer at 100 V for 60–90 minutes at 4°C, or at 30 V overnight. The low voltage overnight approach minimizes protein precipitation and ensures complete transfer.

**Semi-dry transfer** is faster (15–30 minutes at 15–25 V) but requires careful optimization. The small size of histones means they transfer quickly, but they can also pass through the membrane if transfer is prolonged. Use a Tris-glycine buffer without methanol for semi-dry transfer, as methanol is less necessary and can reduce efficiency. Some protocols recommend adding 0.01–0.05% SDS to the transfer buffer to improve elution of histones from the gel, but this can reduce binding to the membrane—test both conditions.

### Ensuring Efficient Transfer of Histones

The most common failure in histone western blots is the complete loss of signal because histones have transferred through the membrane. To prevent this:

1. **Use PVDF membrane** (0.2 µm pore size). PVDF has a higher protein binding capacity (150–200 µg/cm²) than nitrocellulose (80–100 µg/cm²) and binds basic proteins more effectively. Nitrocellulose can work but requires shorter transfer times and is more prone to losing small proteins.
2. **Activate PVDF in methanol** for 15–30 seconds, then equilibrate in transfer buffer before assembly.
3. **Place a second membrane or filter paper behind the primary membrane** during transfer to capture any histones that pass through. This "sandwich" approach allows you to verify transfer completeness by probing the backup membrane.
4. **Verify transfer efficiency** by staining the membrane with Ponceau S (0.1% Ponceau S in 5% acetic acid) after transfer. Histones should appear as distinct bands at 10–17 kDa. If the gel retains significant protein (check by Coomassie staining), increase transfer time or voltage.

## Antibody Selection and Validation

The specificity of [histone modification](/knowledge/molecular-biology/histone-modification) antibodies is the single most important determinant of data quality. Many commercial antibodies cross-react with closely related modifications or recognize unmodified histones.

### Key Considerations for Antibody Specificity

[Histone modification](/knowledge/molecular-biology/histone-modification) antibodies are raised against synthetic peptides carrying the modification of interest. Their specificity depends on:
- **The modification itself**: An antibody against H3K4me3 must not recognize H3K4me2 or H3K4me1. This is particularly challenging for methylation, where the difference is a single methyl group.
- **The surrounding sequence**: Antibodies must recognize the modification in the context of the correct flanking amino acids. For example, an antibody against H3K9me3 should not cross-react with H3K27me3, which shares the ARK(me3)S sequence motif.
- **Species and histone variant**: Some antibodies recognize only specific variants (e.g., H3.3 vs. H3.1) or are raised against human sequences and may not recognize histones from other species.

Always validate antibodies by:
1. **Peptide dot blots**: Spot synthetic peptides (unmodified, mono-, di-, tri-methylated, acetylated, etc.) onto a membrane and probe with the antibody. This confirms specificity against the modification and the methylation state.
2. **Knockout or knockdown controls**: If available, use cells lacking the modifying enzyme (e.g., EZH2 knockout for H3K27me3) to confirm signal loss.
3. **Competition assays**: Pre-incubate the antibody with the immunizing peptide (blocking peptide) to confirm signal abrogation.
4. **Cross-reactivity checks**: Test the antibody against histones from species or tissues where the modification is known to be absent.

### [Positive and Negative Controls](/blog/guides/positive-and-negative-controls-how-to-choose-and-use-them)

Include the following controls in every histone western blot:
- **Positive control**: A sample known to express the modification (e.g., HeLa cells treated with the HDAC inhibitor TSA for acetylation, or cells with constitutive H3K4me3 at active promoters).
- **Negative control**: A sample where the modification is absent or reduced (e.g., cells treated with a methyltransferase inhibitor, or a knockout cell line).
- **Modification-specific vs. pan-histone antibodies**: Use a pan-histone antibody (e.g., anti-total H3, anti-total H4) alongside the modification-specific antibody. This allows normalization and confirms equal loading. Pan-histone antibodies should recognize the histone regardless of PTM status—verify this by testing against hyperacetylated and hypoacetylated samples.

## Blocking, Incubation, and Detection

Optimizing blocking and incubation conditions reduces background and enhances signal-to-noise ratio, which is essential for detecting low-abundance modifications.

### Blocking Agents and Conditions

Block with 5% non-fat dry milk (w/v) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour at room temperature, or overnight at 4°C. However, milk contains casein, which is a phosphoprotein and can cause high background with phospho-specific antibodies. For phospho-histone antibodies, use 5% bovine serum albumin (BSA) in TBST instead. BSA is also preferred for acetylation-specific antibodies, as milk can contain acetylated proteins.

Blocking time should be sufficient to saturate non-specific binding sites but not so long that it strips the antigen from the membrane. One hour at room temperature is typically adequate. For high-background antibodies, extend blocking to 2 hours or overnight at 4°C.

### Signal Detection and Quantification

**Primary antibody incubation**: Dilute the primary antibody in blocking buffer (or TBST with 1% BSA) according to the manufacturer's recommendation, typically 1:1,000 to 1:10,000 for histone antibodies. Incubate overnight at 4°C with gentle agitation. Overnight incubation improves signal-to-noise ratio compared to 1–2 hours at room temperature.

**Secondary antibody incubation**: Use HRP-conjugated secondary antibodies at 1:5,000 to 1:20,000 in blocking buffer for 1 hour at room temperature. For multiplexing, use fluorescent secondary antibodies (e.g., IRDye 680/800) and image on a LI-COR Odyssey or similar system. Fluorescent detection offers a wider linear range and allows simultaneous detection of two targets (e.g., modified H3 and total H3) on the same membrane.

**Washing**: After each antibody incubation, wash the membrane 3–5 times for 5–10 minutes each in TBST. Insufficient washing is a common cause of high background.

**Detection**: For HRP-based detection, use enhanced chemiluminescence (ECL) substrate. Histone signals are typically strong, so a standard ECL substrate is sufficient. If the signal is weak, switch to a more sensitive ECL substrate (e.g., SuperSignal West Femto). Expose the membrane to film or a digital imager. For quantification, ensure that the signal is within the linear range of the detector—avoid saturated bands. Use a series of exposures (e.g., 10 seconds, 30 seconds, 1 minute, 5 minutes) to capture both weak and strong signals.

**Quantification**: Densitometry using ImageJ or similar software. Normalize the modification-specific signal to the total histone signal (e.g., H3K4me3/H3) or to a loading control. For fluorescent detection, use the software's integrated intensity function. Always report the normalized values, not raw intensities.

## Common Pitfalls and Troubleshooting

Even experienced researchers encounter failures in histone western blots. The following are the most frequent issues and their solutions.

### High Background and Non-Specific Bands

**Problem**: The entire lane is smeared, or multiple bands appear that do not correspond to the expected histone molecular weight.

**Causes and solutions**:
- **Insufficient blocking**: Increase blocking time or switch to a different blocking agent (e.g., BSA instead of milk).
- **Antibody concentration too high**: Titrate the primary antibody down (e.g., 1:5,000 instead of 1:1,000). High antibody concentrations often produce non-specific bands.
- **Secondary antibody cross-reactivity**: If using a secondary antibody raised against the same species as the primary, it may recognize endogenous immunoglobulins. Use a species-specific secondary antibody and include a no-primary control.
- **Histone degradation**: Degraded histones produce a ladder of lower-molecular-weight bands. Check protease inhibitor freshness and work quickly on ice.
- **Modified histones with altered mobility**: Acetylated histones migrate more slowly (higher apparent molecular weight) than unmodified forms. Multiple bands may represent different acetylation states. This is expected and informative, but if you are probing for a single modification, ensure your antibody is specific and that you are not seeing cross-reactivity with other modifications.

### Weak or No Signal

**Problem**: No bands are visible, or the signal is too weak to quantify.

**Causes and solutions**:
- **Histones did not transfer**: Check the membrane with Ponceau S. If no bands are visible, increase transfer time or voltage, reduce methanol concentration, or add 0.01% SDS to the transfer buffer.
- **Histones passed through the membrane**: If the backup membrane shows signal, reduce transfer time or use a smaller pore size (0.2 µm instead of 0.45 µm).
- **Antibody failure**: Validate the antibody with a positive control. If the positive control fails, the antibody may be expired or non-functional.
- **Insufficient antigen**: Increase the amount of histone protein loaded (up to 20 µg per lane).
- **Detection reagent failure**: Check the ECL substrate by exposing a known positive membrane. Ensure the HRP-conjugated secondary antibody is fresh.
- **Membrane over-blocked**: Excessive blocking can mask the antigen. Reduce blocking time or use a lower concentration of blocking agent.

### Inconsistent Results Across Experiments

**Problem**: The same sample gives different band intensities or patterns in replicate experiments.

**Causes and solutions**:
- **Sample degradation**: PTMs, especially phosphorylation and acetylation, are labile. Ensure that inhibitors are fresh and that samples are stored at −80°C in small aliquots to avoid freeze-thaw cycles.
- **Transfer variability**: Semi-dry transfer is more variable than wet transfer. Standardize transfer conditions and use a transfer apparatus with even pressure.
- **Gel-to-gel variability**: Prepare gels fresh and use the same batch of reagents. For precast gels, use the same brand and lot.
- **Antibody lot variability**: Different lots of the same antibody can have different specificities. Validate each new lot and store antibodies at 4°C (not frozen) to prevent degradation.
- **Exposure variability**: For chemiluminescence, the signal decays over time. Expose membranes at consistent time points after ECL addition.

## Summary and Best Practices

A successful histone western blot requires attention to the unique properties of histones at every step. The following checklist summarizes the key practices:

1. **Extract histones** using acid extraction or nuclear isolation with fresh protease, deacetylase, and phosphatase inhibitors.
2. **Quantify** histone samples by Coomassie-stained gel densitometry, not A₂₈₀.
3. **Separate** histones on a 16% Tris-Tricine gel or a 12% Bis-Tris gel with MES buffer. Add urea if resolving modified forms.
4. **Transfer** to 0.2 µm PVDF membrane using wet transfer at 100 V for 60–90 minutes in buffer with 10% methanol. Verify with Ponceau S.
5. **Validate** all histone modification antibodies by peptide dot blots and positive/negative controls.
6. **Block** with 5% BSA for phospho- or acetyl-specific antibodies; 5% milk is acceptable for others.
7. **Incubate** with primary antibody overnight at 4°C, wash thoroughly, and detect with ECL or fluorescence.
8. **Quantify** within the linear range and normalize to total histone levels.

## Frequently Asked Questions

### What is the best protocol for histone western blot?

The best protocol depends on your specific modification of interest, but a robust general approach is: acid-extract histones from 1–5 × 10⁶ cells using 0.2 M HCl, precipitate with 25% TCA, wash with acetone, and resuspend in Laemmli buffer. Separate 1–10 µg on a 16% Tris-Tricine gel, transfer to 0.2 µm PVDF membrane by wet transfer at 100 V for 90 minutes in Tris-glycine buffer with 10% methanol. Block with 5% BSA in TBST, incubate with validated primary antibody overnight at 4°C, and detect with HRP-conjugated secondary and ECL. Always include a total histone control (e.g., anti-H3) for normalization.

### Why do I see multiple bands in my histone western blot?

Multiple bands can arise from (1) histone degradation producing lower-molecular-weight fragments, (2) PTM-induced mobility shifts (e.g., hyperacetylated H4 migrates more slowly), (3) histone variants with different molecular weights, or (4) antibody cross-reactivity with other histones or unrelated proteins. To distinguish these, run a Coomassie-stained gel to check histone integrity, use a pan-histone antibody to identify the expected band pattern, and validate your modification-specific antibody with peptide competition.

### How can I reduce high background in histone western blots?

High background is usually caused by excessive antibody concentration, insufficient blocking, or inadequate washing. Titrate the primary antibody down (test 1:1,000, 1:5,000, 1:10,000), increase blocking time to 2 hours or overnight, and wash 5 times for 10 minutes each. For phospho-specific antibodies, switch from milk to BSA blocking. If background persists, use a more dilute secondary antibody or switch to fluorescent detection.

### What is the recommended transfer buffer for histone western blots?

The standard transfer buffer is 25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3. Use 10% methanol rather than 20% to improve transfer efficiency for small proteins. For semi-dry transfer, methanol can be omitted, but wet transfer is more reliable. Adding 0.01–0.05% SDS can improve elution from the gel but may reduce membrane binding—test both conditions.

### Can I use nitrocellulose membrane for histone western blots?

Yes, but with caution. Nitrocellulose has lower protein binding capacity than PVDF and is more prone to losing small basic proteins during washing. If using nitrocellulose, use a 0.2 µm pore size, reduce transfer time to prevent pass-through, and avoid harsh washing conditions. PVDF is strongly recommended for histones due to its higher binding capacity and mechanical strength.

### How do I choose the right histone modification antibody?

Select antibodies that have been validated by the manufacturer for western blotting and for the specific modification state (e.g., H3K4me3, not just "H3K4"). Check the datasheet for cross-reactivity data against other methylation states and modifications. Independently validate by peptide dot blots and with positive/negative cell lines. Prefer antibodies raised against the modification in the correct sequence context (e.g., H3K9me3 antibodies should be raised against the ARTKQTARK(me3)STGG sequence, not a generic methyl-lysine peptide).

### Why is my histone signal weak or absent?

Weak or absent signal is most commonly due to inefficient transfer (histones remain in the gel or pass through the membrane), degraded samples, or failed antibodies. Check the membrane with Ponceau S after transfer—if no bands are visible, the transfer failed. If bands are visible but no signal, validate the antibody with a positive control. If the positive control works, increase the amount of histone loaded or use a more sensitive ECL substrate.

### What are common mistakes in histone western blotting?

Common mistakes include: (1) using standard Laemmli gels that poorly resolve histones, (2) overloading the gel, causing smearing, (3) using transfer conditions optimized for larger proteins, leading to histone loss, (4) failing to include deacetylase and phosphatase inhibitors, resulting in loss of labile modifications, (5) using unvalidated antibodies that cross-react, and (6) normalizing to total histone when total histone levels change between conditions.

## Key Takeaways

- Histones require specialized conditions at every step—acid extraction, Tris-Tricine or Bis-Tris gels, PVDF membranes, and validated modification-specific antibodies—because they are small, basic, and highly abundant.
- Always include protease, deacetylase, and phosphatase inhibitors during extraction to preserve PTMs, especially when studying acetylation or phosphorylation.
- Use 0.2 µm PVDF membrane and wet transfer with 10% methanol to ensure efficient retention of histones; verify transfer with Ponceau S staining.
- Validate every histone modification antibody by peptide dot blots and positive/negative controls; methylation-state specificity is particularly critical.
- Normalize modification signals to total histone levels (e.g., H3) only when total histone levels are unchanged; otherwise, use total protein staining.
- Troubleshoot systematically: check transfer efficiency first, then antibody specificity, then blocking and washing conditions.
- For reproducible results, standardize gel preparation, transfer conditions, and exposure times, and store samples in small aliquots at −80°C to avoid freeze-thaw degradation.

## Further Reading

- Feng Y et al. *Identifying new sperm [Western blot loading controls](/blog/guides/western-blot-loading-controls)*. Andrologia. 2021. [PubMed 34478154](https://doi.org/10.1111/and.14226)
- Zhang K et al. *A mass spectrometric "Western blot" to evaluate the correlations between [histone methylation](/knowledge/molecular-biology/histone-methylation) and [histone acetylation](/knowledge/molecular-biology/histone-acetylation)*. Proteomics. 2004. [PubMed 15378694](https://doi.org/10.1002/pmic.200400819)
- Beyer M et al. *How to Distinguish Between the Activity of HDAC1-3 and HDAC6 with Western Blot*. Methods in molecular biology (Clifton, N.J.). 2017. [PubMed 27761834](https://doi.org/10.1007/978-1-4939-6527-4_26)
- Devi BJ, Schneeweiss FH, Sharan RN. *Negative correlation between poly-ADP-ribosylation of spleen cell histone proteins and initial duration of dimethylnitrosamine exposure to mice in vivo measured by Western blot immunoprobe assay: a possible biomarker for cancer detection*. Cancer detection and prevention. 2005. [PubMed 15734220](https://doi.org/10.1016/j.cdp.2004.10.004)

## Related Clinical & Scientific Guides

* [MAPK Pathway: Mechanism, Function, and Clinical Relevance](/knowledge/molecular-biology/mapk-pathway)
* [Mammalian Cell Culture Bioreactors: A Practical Guide](/knowledge/molecular-biology/mammalian-cell-culture-bioreactor)
* [Nucleotide Formation: Biosynthesis and Assembly of DNA/RNA Building Blocks](/knowledge/molecular-biology/nucleotide-formation)