POU3F3 Antibody

What is POU3F3 and what are its key biological functions?

POU3F3 (also known as Brain-1, BRN1, OTF8, Oct-8) belongs to the class III POU family of transcription factors that are predominantly expressed in the central nervous system . The POU domain in these proteins is required for high-affinity binding to octamer DNA sequences (5'-ATTTGCAT-3') .

At the molecular level, POU3F3:

• Acts synergistically with SOX11 and SOX4 transcription factors

• Plays a critical role in neuronal development

• Is implicated in enhancer activity at the embryonic met-mesencephalic junction

• Has demonstrated importance in kidney development, particularly in the thick ascending limb (TAL) of the loop of Henle

Research with Pou3f3 mutant mice has revealed its involvement in regulating nephron numbers in the kidney, with homozygous mutants displaying smaller kidney volumes, decreased nephron numbers, and reduced TAL volumes .

When selecting a POU3F3 antibody, researchers should consider several technical aspects:

• Host Species: Available as rabbit polyclonal , goat polyclonal , and mouse monoclonal antibodies

• Target Epitope: Different antibodies target different regions:

  • HPA056039: Immunogen sequence QGPDVKGGAGRDDLHAGTALHHR

  • PA5-64311: Immunogen sequence GGAQSLVHPGLVRGDTPELAEHHHH

  • A84814: Immunogen sequence C-HMLSHAHQWVTAL (internal region of human POU3F3)

• Validation Method: Consider antibodies with enhanced validation such as:

  • Orthogonal RNAseq validation

  • Validation in multiple species or applications

• Molecular Weight: The calculated molecular weight of POU3F3 is approximately 50 kDa, but observed weight in Western blot can be 66 kDa , which is important to note when analyzing results

• Applications: Select based on your specific application needs, as validation breadth varies between products

How do POU3F3 antibodies perform across different species?

POU3F3 antibodies show varying degrees of cross-reactivity across species, which is important to consider for comparative studies:

When working with model organisms, researchers should note that human POU3F3 shares high sequence homology with mouse (P31361) and rat (Q63262) orthologs , which explains the cross-reactivity of many antibodies.

For cross-species studies, antibodies targeting highly conserved regions of the protein (particularly within the POU domain) will likely show better cross-reactivity.

What are the optimal protocols for POU3F3 detection in immunohistochemistry?

For optimal POU3F3 detection in immunohistochemistry, the following protocol parameters are recommended:

Antigen Retrieval:

• Primary method: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Antibody Dilutions:

• For standard IHC: 1:20-1:200 (product 18999-1-AP)

• For IHC with enhanced sensitivity: 1:200-1:500 (product HPA067151)

Visualizing Systems:

For brain tissue samples, POU3F3 antibodies have been successfully used with both chromogenic (peroxidase-based) and fluorescent secondary detection systems .

Tissue-Specific Considerations:

When examining POU3F3 in brain tissues, researchers should note the nuclear localization pattern typical of transcription factors . For kidney studies, pay particular attention to the thick ascending limb of the loop of Henle where POU3F3 plays a critical developmental role .

Published immunohistochemistry images show strong nuclear staining in neuronal cells in human brain tissue samples , which can serve as a positive control pattern.

How can researchers validate the specificity of POU3F3 antibodies?

Validating antibody specificity is critical for reliable results. For POU3F3 antibodies, consider these validation approaches:

• RNA Interference: Compare staining patterns between control and POU3F3-knockdown samples. The specific signal should be significantly reduced in knockdown samples.

• Genetic Models: Utilize tissue from Pou3f3 mutant mice, such as the Pou3f3L423P mouse line, which harbors a T→C point mutation leading to an amino acid exchange from leucine to proline in the conserved homeobox domain .

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific staining should be blocked by this competition.

• Orthogonal Validation: Compare protein expression patterns with mRNA expression data. The HPA067151 antibody, for example, has been validated using orthogonal RNAseq approaches .

• Multiple Antibody Concordance: Use multiple antibodies targeting different epitopes of POU3F3 to confirm consistent staining patterns.

• Expected Expression Pattern: Verify that staining matches the known tissue distribution of POU3F3, with strong expression in the central nervous system and developing kidney .

Successful validation should demonstrate specific nuclear localization consistent with POU3F3's function as a transcription factor.

What are the differences between polyclonal and monoclonal POU3F3 antibodies for research applications?

When choosing between polyclonal and monoclonal POU3F3 antibodies, researchers should consider these key differences:

For POU3F3 research specifically:

• Polyclonal antibodies like 18999-1-AP have been cited in multiple publications and validated across several applications

• The Atlas Antibodies monoclonal antibody (AMAB92023) represents a newer option for researchers seeking higher reproducibility

For studies requiring dual-labeling with other rabbit antibodies, the goat polyclonal (A84814) or mouse monoclonal options may be preferable to avoid species cross-reactivity .

What is known about POU3F3's role in kidney development based on current research?

POU3F3 plays a critical role in kidney development, particularly affecting nephron number and the thick ascending limb (TAL) of the loop of Henle. Key findings from studies using Pou3f3 mutant mice include:

• Morphological Abnormalities: Homozygous Pou3f3L423P mutant mice display:

  • Significantly smaller kidney volumes

  • Decreased nephron numbers

  • Smaller TAL volumes

  • Lower volume densities of the TAL in the kidney

• Functional Consequences:

  • Aged homozygous mutants show increased serum urea concentrations

  • Reduced specific urine gravity

  • No evidence of glomerular dysfunction

• Histological Observations:

  • No histological or ultrastructural lesions of TAL cells or glomerular cells were observed

  • The primary effect appears to be on organ development rather than cellular pathology

These findings confirm POU3F3's role in development and function of the TAL and provide evidence for its involvement in regulating nephron number. The Pou3f3L423P mutant mouse model represents a valuable research tool for nephrological studies examining the consequences of congenital low nephron numbers .

How should researchers design experiments to study POU3F3 in cell culture systems?

When designing experiments to study POU3F3 in cell culture systems, researchers should consider:

Cell Line Selection:

Based on validated antibody data, these cell lines have been successfully used for POU3F3 research:

• SH-SY5Y (human neuroblastoma cells)

• Neuro-2a (mouse neuroblastoma cells)

• HEK-293 (human embryonic kidney cells)

Experimental Controls:

• Positive Controls: Use brain tissue lysates as positive controls for Western blot

• Negative Controls:

  • For flow cytometry: Include unimmunized IgG controls

  • For immunofluorescence: Include secondary-only controls

Protocol Recommendations:

For Flow Cytometry:

• Fix cells with paraformaldehyde

• Permeabilize with 0.5% Triton

• Primary antibody incubation: 1 hour (10μg/ml)

• Use appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488)

For Immunofluorescence:

• Fix cells with paraformaldehyde

• Permeabilize with 0.15% Triton

• Use antibody at 10μg/ml concentration

For Western Blot:

• Expected molecular weight: Calculated 50 kDa, but often observed at 66 kDa

• Recommended dilution range: 1:500-1:2000

Functional Studies:

To investigate POU3F3's transcriptional activity, consider reporter assays using the octamer motif (5'-ATTTGCAT-3') which POU3F3 binds to with high affinity .

How can researchers troubleshoot common problems with POU3F3 antibody detection?

When troubleshooting POU3F3 antibody detection issues, consider these common problems and solutions:

Weak or No Signal in Western Blot:

• Problem: POU3F3 may be expressed at low levels in some tissues

• Solutions:

  • Increase protein loading (50-100μg recommended)

  • Use more sensitive detection systems (ECL Plus/Prime)

  • Try longer exposure times

  • Consider antibodies observing the 66 kDa band rather than the calculated 50 kDa

High Background in Immunohistochemistry:

• Problem: Nonspecific binding, especially in brain tissue

• Solutions:

  • Increase blocking time (≥1 hour with 5% BSA or serum)

  • Optimize antibody dilution (start with 1:100 and titrate)

  • Test alternative antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

Inconsistent Results Across Different Lots:

• Problem: Batch-to-batch variation, especially with polyclonal antibodies

• Solutions:

  • Consider switching to monoclonal antibodies for better consistency

  • Validate each new lot against a reference sample

  • Include positive control tissues (human or mouse brain) in each experiment

Cross-Reactivity Issues:

• Problem: Antibody detecting non-target proteins

• Solutions:

  • Verify specificity through knockout/knockdown controls

  • Consider antibodies validated through orthogonal RNAseq

  • Use antibodies with published validation data in your application

Species Cross-Reactivity Failure:

• Problem: Antibody not working in expected species

• Solution: Choose antibodies with documented cross-reactivity or those targeting highly conserved epitopes

What recent advances have been made in understanding POU3F3 function in disease models?

Recent research has expanded our understanding of POU3F3's role in various disease states:

Neurological Disorders:

• POU3F3 has been implicated in neuronal development pathways, acting synergistically with SOX11 and SOX4

• Research suggests potential linkages with neuronitis and cerebritis

Renal Pathology:

• Missense mutations in POU3F3 (specifically L423P) affect kidney development

• Homozygous mutant mice display phenotypes that provide insight into congenital nephron reduction

• POU3F3 has been studied in the context of polycystic kidney disease, where PKD1 and PKD2 genes were inactivated by gene editing

Cancer Research:

• Linc-POU3F3 (long intergenic non-coding RNA associated with POU3F3) has been investigated for its role in promoting cell proliferation in gastric cancer

• This suggests potential roles for POU3F3-related regulatory elements in cancer progression

Researchers focusing on these disease areas should consider the specific antibody requirements for their model systems, including species reactivity and application optimization for the particular tissue type.

What considerations are important when using POU3F3 antibodies in immunoprecipitation experiments?

When planning immunoprecipitation (IP) experiments with POU3F3 antibodies, researchers should consider:

Antibody Selection:

• While IP applications aren't explicitly listed in the search results, antibodies purified by antigen affinity chromatography (such as 18999-1-AP and A84814) may be suitable candidates

• Consider using antibodies with proven specificity in Western blot applications as a starting point

Experimental Design:

• Lysate Preparation:

  • For nuclear proteins like POU3F3, use nuclear extraction protocols

  • Include protease inhibitors to prevent degradation

  • Consider crosslinking approaches for studying DNA-protein interactions

• Controls:

  • Input sample (pre-IP lysate)

  • Negative control IP with isotype-matched IgG

  • If available, lysate from POU3F3-knockout or knockdown cells

• Technical Considerations:

  • Antibody amount: Start with 2-5μg per 500μg protein lysate

  • Incubation: Overnight at 4°C with gentle rotation

  • Washing: Use stringent conditions to reduce background

Applications Beyond Standard IP:

• Chromatin Immunoprecipitation (ChIP): Given POU3F3's role as a transcription factor that binds to octamer DNA sequences (5'-ATTTGCAT-3'), ChIP experiments could identify genomic binding sites

• Co-IP: Investigate protein interactions with known partners (SOX11, SOX4) or discover new ones