SAL2 Antibody

What is SALL2 and what biological functions does it serve?

SALL2 (Sal-like protein 2) is a transcription factor that plays an important role in eye development before, during, and after optic fissure closure. This protein functions as a p53-independent regulator of p21 and BAX, serving as a regulator of cell growth and survival in specific cell types. Human SALL2 is primarily expressed in brain, heart, kidney, and pancreas tissues. The protein is predominantly localized to the nucleus, consistent with its role as a transcription factor .

At the molecular level, SALL2 belongs to the spalt-like family of zinc finger proteins, sharing homology with Drosophila region-specific homeotic gene spalt (sal). Its full molecular structure includes 1007 amino acids with a calculated molecular weight of 105 kDa, though it typically appears at approximately 130 kDa when detected in Western blot applications .

What types of SALL2 antibodies are available for research applications?

Current research-grade SALL2 antibody options include:

These antibodies are typically supplied in liquid form, formulated in PBS with stabilizers such as glycerol (50%), BSA (0.5%), and sodium azide (0.02%) as a preservative. The antibodies are affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography to ensure specificity .

It's important to note that all commercially available SALL2 antibodies are designated strictly for research use only (RUO) and must not be used in diagnostic or therapeutic applications .

How should I validate a SALL2 antibody for my specific research application?

Validation of a SALL2 antibody requires a systematic approach specific to your experimental conditions:

• Initial verification: Begin by confirming antibody integrity through SDS-PAGE to ensure no degradation has occurred during shipping or storage.

• Specificity testing: Use positive controls (tissues/cells known to express SALL2 such as Jurkat cells or HEK-293 cells) and negative controls (knockout cells or tissues with confirmed absence of SALL2) .

• Application-specific validation: Validation must be performed for each specific application. For Western blot, test different dilutions (ranging from 1:500 to 1:2000) to determine optimal signal-to-noise ratio .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other SAL family proteins by testing in samples containing varying concentrations of target and non-target proteins.

• Reproducibility testing: Perform replicate experiments to ensure consistent results across different days and experimental conditions.

Remember that antibody validation is not a one-time process but should be repeated when changing experimental parameters such as cell types, tissue sources, or detection methods .

What are the optimal conditions for using SALL2 antibody in Western blot applications?

For optimal Western blot results with SALL2 antibody:

• Sample preparation: Extract proteins using lysis buffers containing protease inhibitors to prevent degradation of SALL2, which typically appears at approximately 130 kDa.

• Protein loading: Load 20-50 μg of total protein per lane, with higher amounts potentially needed for tissues with lower SALL2 expression.

• Dilution optimization: Start with a dilution range of 1:500-1:2000 and optimize based on signal strength and background .

• Blocking conditions: Use 5% non-fat dry milk or 3-5% BSA in TBST for blocking, typically for 1 hour at room temperature.

• Primary antibody incubation: Incubate with diluted SALL2 antibody overnight at 4°C for optimal binding.

• Washing steps: Perform at least 3-5 washes with TBST (5-10 minutes each) after primary and secondary antibody incubations to reduce background.

• Secondary antibody: Use anti-rabbit IgG conjugated to HRP at 1:5000-1:10000 dilution for 1 hour at room temperature.

• Expected molecular weight: Verify that the detected band appears at approximately 130 kDa, which is the observed molecular weight for SALL2 despite its calculated weight of 105 kDa .

How can SALL2 antibody be optimized for dual-recognition assays such as co-immunoprecipitation or sandwich ELISA?

For dual-recognition assays involving SALL2:

• Epitope selection: When designing a sandwich assay, select antibodies targeting different epitopes of SALL2. Current commercial options include antibodies targeting the 956-1006 amino acid region and others using fusion protein immunogens .

• Capture antibody optimization: For sandwich ELISA, coat plates with the SALL2 antibody at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C. Polyclonal antibodies often work well as capture antibodies due to their recognition of multiple epitopes.

• Detection antibody considerations: For the detection step, consider using a more specific monoclonal antibody (if available) or a differently labeled polyclonal antibody that targets a distinct epitope.

• Co-immunoprecipitation strategy: When using SALL2 antibody for co-IP:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate antibody with protein A/G beads before adding to lysate

  • Use gentle washing conditions to preserve protein-protein interactions

  • Include appropriate negative controls (IgG from same species)

• Cross-validation: Verify results using reciprocal co-IP approaches when investigating protein-protein interactions with SALL2.

Dual-recognition approaches significantly enhance specificity, allowing for more reliable detection of SALL2 even when using antibodies with moderate individual specificity .

What approaches can resolve discrepancies in SALL2 detection between different experimental techniques?

When facing inconsistent results in SALL2 detection:

• Protein size discrepancy analysis: SALL2 has a calculated molecular weight of 105 kDa but often appears at 130 kDa in Western blots . This discrepancy may be due to:

  • Post-translational modifications

  • Splice variants

  • Incomplete denaturation

  • Migration anomalies due to protein structure

• Expression level verification: Confirm SALL2 expression using orthogonal methods:

  • RT-qPCR to quantify mRNA levels

  • Mass spectrometry to confirm protein identity

  • Multiple antibodies targeting different epitopes

• Technique-specific considerations:

  • For Western blot: Modify transfer conditions for high molecular weight proteins

  • For IHC/ICC: Try different fixation methods and antigen retrieval techniques

  • For ELISA: Adjust coating conditions and blocking reagents

• Sample preparation impact: Different lysis buffers can affect epitope availability:

  • RIPA buffer may destroy some conformational epitopes

  • NP-40 or Triton X-100 based buffers might better preserve protein structure

  • For nuclear proteins like SALL2, ensure proper nuclear extraction techniques

• Control experiments: Always include proper controls to interpret discrepancies:

  • Positive controls (Jurkat cells, HEK-293 cells)

  • Negative controls (knockdown/knockout samples)

  • Loading controls for normalization

How can I address weak or absent signal when using SALL2 antibody in Western blot?

When encountering weak or no signal with SALL2 antibody:

• Protein expression verification: Confirm that your sample expresses SALL2. Remember that expression is highest in brain, heart, kidney, and pancreas tissues .

• Sample preparation optimization:

  • Ensure complete protein extraction, particularly for nuclear proteins like SALL2

  • Add phosphatase and protease inhibitors immediately after cell lysis

  • Avoid repeated freeze-thaw cycles of protein samples

• Technical adjustments:

  • Decrease antibody dilution (try 1:500 instead of 1:2000)

  • Increase protein loading (up to 50-75 μg per lane)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced sensitivity detection reagents (e.g., enhanced chemiluminescence)

  • Optimize transfer conditions for high molecular weight proteins

• Antibody quality assessment:

  • Check antibody expiration date and storage conditions

  • Run a dot blot with purified antigen (if available) to verify antibody activity

  • Consider testing a different lot or alternative SALL2 antibody

• Signal enhancement strategies:

  • Use signal enhancing systems (biotin-streptavidin)

  • Consider tyramide signal amplification for immunohistochemistry applications

  • Increase exposure time during imaging (while monitoring background)

What strategies can minimize background or non-specific binding when using SALL2 antibody?

To reduce background and non-specific binding:

• Blocking optimization:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blocking buffers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Antibody dilution adjustments:

  • Prepare antibody in fresh blocking buffer

  • Increase antibody dilution incrementally (1:1000, 1:2000, etc.)

  • Pre-absorb antibody with non-specific proteins from the species of your samples

• Washing protocol enhancement:

  • Increase number of washes (5-6 times)

  • Extend washing time (10-15 minutes per wash)

  • Add higher concentration of detergent (0.1-0.2% Tween-20) in washing buffer

• Sample-specific considerations:

  • For tissues with high endogenous peroxidase activity, include quenching steps

  • For tissue sections, optimize antigen retrieval methods

  • For cell lines, consider fixation method impact on epitope accessibility

• Technical controls:

  • Include a secondary antibody-only control

  • Use isotype control antibody as negative control

  • Block with immunizing peptide to confirm specificity (if available)

How can SALL2 antibody be utilized in studying cancer biology and potential therapeutic targets?

SALL2 has been implicated in cell growth regulation and survival, making it relevant for cancer research:

• Expression profiling:

  • Use SALL2 antibody for Western blot or IHC to analyze expression patterns across cancer types

  • Compare SALL2 levels between tumor and adjacent normal tissues

  • Correlate expression with clinical parameters and patient outcomes

• Functional investigations:

  • Study SALL2 interaction with p21 and BAX in cancer cells using co-IP with SALL2 antibody

  • Investigate SALL2 localization changes during cell cycle progression using ICC/IF

  • Analyze SALL2 binding partners in different cancer contexts through antibody-based pull-down assays

• Therapeutic target assessment:

  • Monitor SALL2 expression changes in response to various treatments

  • Evaluate SALL2 as a biomarker for treatment response

  • Study post-translational modifications of SALL2 that might be targeted therapeutically

• Technical considerations for cancer samples:

  • Optimize antibody concentration for tissue microarrays

  • Establish appropriate positive and negative controls for each cancer type

  • Consider tumor heterogeneity when interpreting staining patterns

SALL2's role as a p53-independent regulator of p21 and BAX suggests potential involvement in apoptotic pathways and cell cycle regulation, which are critical processes in cancer development and treatment response .

What are the considerations for using SALL2 antibody in multi-parameter flow cytometry or mass cytometry?

For incorporating SALL2 antibody into multi-parameter cytometry:

• Antibody modification requirements:

  • For flow cytometry: Conjugate SALL2 antibody to appropriate fluorophores (e.g., FITC, PE, APC)

  • For mass cytometry (CyTOF): Conjugate with rare earth metals

• Cell preparation protocol:

  • Optimize fixation and permeabilization conditions for nuclear protein detection

  • Consider methanol-based permeabilization for optimal nuclear antigen access

  • Test different fixation durations to balance epitope preservation and cell integrity

• Panel design considerations:

  • Select compatible fluorophores that minimize spectral overlap

  • Include appropriate compensation controls

  • Place SALL2 detection in a channel with appropriate sensitivity for expected expression levels

• Validation approaches:

  • Confirm specificity using SALL2 knockdown/knockout cells

  • Compare flow cytometry results with Western blot data for consistency

  • Use fluorescence-minus-one (FMO) controls to set proper gates

• Data analysis recommendations:

  • Consider SALL2 expression as a continuous variable rather than positive/negative

  • Correlate SALL2 levels with other transcription factors or cell cycle markers

  • Use dimensionality reduction techniques (tSNE, UMAP) for high-parameter data visualization

SALL2's nuclear localization presents special challenges for cytometry applications, requiring careful optimization of permeabilization protocols to allow antibody access while preserving cellular structure and other epitopes of interest.

How can AI and machine learning approaches enhance the specificity prediction and application optimization of SALL2 antibodies?

Emerging computational approaches offer new possibilities for SALL2 antibody research:

• Epitope prediction refinement:

  • Machine learning algorithms can predict optimal epitopes for antibody generation

  • Structural modeling can identify surface-exposed regions of SALL2 most suitable for antibody targeting

  • Analysis of protein sequence conservation can identify species-specific versus conserved epitopes

• Specificity prediction models:

  • Deep learning approaches can analyze antibody sequences to predict binding specificity

  • Models trained on large antibody datasets can predict cross-reactivity potential

  • Computational docking can simulate antibody-SALL2 interactions

• Experimental design optimization:

  • AI-driven experimental design can identify optimal conditions for SALL2 antibody applications

  • Automated image analysis can improve quantification of immunohistochemistry results

  • Machine learning can help identify patterns in large-scale antibody validation datasets

Recent research has demonstrated success in developing sequence-based deep learning models to predict antibody specificity, suggesting similar approaches could be applied to enhance SALL2 antibody applications .

What validation standards should researchers apply when evaluating new SALL2 antibodies or novel applications of existing antibodies?

Comprehensive validation of SALL2 antibodies should include:

• Foundational validation steps:

  • Western blot showing single band at expected molecular weight (130 kDa)

  • Positive controls using cell lines with confirmed SALL2 expression (Jurkat, HEK-293)

  • Negative controls using SALL2 knockdown/knockout samples

• Advanced specificity assessments:

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays using the immunizing peptide

  • Testing across multiple cell types and tissues with varying SALL2 expression levels

• Application-specific validation:

  • For each new application, perform dedicated validation experiments

  • Document optimal conditions for dilution, incubation time, and buffer composition

  • Evaluate batch-to-batch consistency when using the antibody over time

• Reporting standards:

  • Provide complete validation data when publishing

  • Include detailed methods sections describing antibody source, catalog number, lot number, and validation procedures

  • Share raw validation data when possible to support reproducibility

• Orthogonal method confirmation:

  • Confirm key findings using alternative detection methods

  • Correlate protein detection with mRNA expression data

  • Use genetic approaches (overexpression, knockdown) to verify antibody specificity

Proper validation requires demonstrating the antibody's selectivity in the specific application, experimental conditions, and relevant concentration ranges of both target and potential off-target proteins .