OCT2 Antibody

What is OCT2 and why are there two distinct proteins with this name?

OCT2 refers to two different proteins that share the same abbreviation but have entirely distinct functions:

• SLC22A2 (OCT2): A member of the SLC22A superfamily of transporters that are polyspecific and involved in the absorption and excretion of various endogenous and exogenous compounds. This family consists of 18 genes and includes organic cation transporters (OCTs), organic cation/carnitine transporters (OCTNs), and organic anion transporters (OATs) .

• POU2F2 (OCT2): A transcription factor of the POU homeo-domain family that binds to immunoglobulin gene octamer sites, regulating B-cell-specific genes. It functions as a marker of B-cell lineage and differentiation .

This dual nomenclature often creates confusion in research, so clarifying which OCT2 protein is being studied is essential for experimental design and interpretation.

What tissues express each type of OCT2?

SLC22A2 (Transporter) Expression:

• Primarily expressed in proximal tubules of the kidney, where it plays a critical role in cation transport and drug excretion

• Also expressed in neurons, particularly in the rat medial septum nucleus as demonstrated by immunohistochemical analysis

• Can be detected in mouse kidney lysates and rat kidney lysates via Western blot

POU2F2 (Transcription Factor) Expression:

• Highly expressed in germinal center B-cells, mantle B-cells, monocytoid B-cells, and plasma cells

• Increased expression observed in various lymphomas including mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, plasmacytoma, Burkitt lymphoma, diffuse large cell lymphoma, diffuse large B-cell lymphoma, and Hodgkin lymphoma

• Low-level expression in pre-B, T-cell, myelomonocytic, and epithelial cell lines

• Detected in NIH/3T3 cells and mouse ovary tissue

What are the primary applications for OCT2 antibodies?

OCT2 antibodies are utilized in multiple experimental applications, with variations depending on which OCT2 protein is being studied:

This range of applications makes OCT2 antibodies versatile tools for investigating protein expression, localization, and function in various research contexts .

How can I validate the specificity of my OCT2 antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For OCT2 antibodies, consider these validation approaches:

For SLC22A2 (Transporter) Antibodies:

• Use blocking peptides: Pre-incubation of the antibody with SLC22A2 blocking peptide (such as BLP-CT020) should suppress staining in immunohistochemistry and bands in Western blot, as demonstrated in both mouse kidney and rat brain samples

• Use positive control tissues: Mouse or rat kidney lysates serve as excellent positive controls for Western blot validation

• Perform immunohistochemistry on kidney sections, where OCT2 immunoreactivity should appear in proximal tubules

For POU2F2 (Transcription Factor) Antibodies:

• Use established positive control cell lines like NIH/3T3 cells or B-cell lines known to express high levels of OCT2

• Test antibody performance in human lymphoma tissue, where OCT2 expression is well-characterized

• Validate signal at the expected molecular weight (calculated MW is 44 kDa, observed MW is approximately 55 kDa for POU2F2)

For both proteins, knockout or knockdown models provide gold-standard validation but may not be readily available in all research settings.

What is the optimal antigen retrieval method for OCT2 antibodies in immunohistochemistry?

Antigen retrieval methods differ depending on the specific OCT2 protein and tissue preparation:

For SLC22A2 (Transporter):

• For formalin-fixed paraffin-embedded (FFPE) mouse kidney sections, heat-induced epitope retrieval with citrate buffer at pH 6.0 has been validated

• For perfusion-fixed frozen rat brain sections, antigen retrieval may not be necessary

For POU2F2 (Transcription Factor):

• For human lymphoma tissue, the recommended method is antigen retrieval with TE buffer at pH 9.0

• As an alternative, citrate buffer at pH 6.0 can also be used, though it may yield different results in terms of signal intensity

The choice of antigen retrieval method should be empirically determined for each tissue type and fixation method, particularly when working with different species or tissue preparations.

How do I troubleshoot non-specific binding with OCT2 antibodies?

Non-specific binding can compromise experimental results. Here are strategies to address this issue:

For Western Blot Applications:

• Optimize antibody concentration: For SLC22A2, a 1:200 dilution has been validated ; for POU2F2, a 1:500-1:1000 dilution is recommended

• Use appropriate blocking conditions: 5% non-fat dry milk or bovine serum albumin (BSA) in TBST or PBST

• Increase washing steps and duration to remove unbound antibody

• Include a positive control (kidney lysate for SLC22A2; NIH/3T3 cells for POU2F2)

• For SLC22A2, confirm specificity using the blocking peptide BLP-CT020

For Immunohistochemistry Applications:

• Optimize antibody dilution: For SLC22A2, 1:100 for FFPE sections and 1:200 for frozen sections has been validated ; for POU2F2, a broader range of 1:20-1:200 is suggested

• Perform antigen retrieval appropriately for each tissue and fixation method

• Include adequate blocking of endogenous peroxidases (if using HRP-conjugated secondary antibodies)

• Use proper isotype controls: For POU2F2 monoclonal antibodies, an IgG1 kappa control should be used

• For SLC22A2, include a blocking peptide control to demonstrate specificity

What are the considerations for selecting between monoclonal and polyclonal OCT2 antibodies?

The choice between monoclonal and polyclonal antibodies impacts experimental outcomes:

Select the appropriate antibody type based on your experimental needs. For highly specific detection of a particular epitope, monoclonal antibodies may be preferred. For applications where signal strength is paramount or when protein conformation might be altered, polyclonal antibodies offer advantages.

What are the recommended dilutions and conditions for Western blot using OCT2 antibodies?

Optimal working conditions for Western blot vary depending on the specific OCT2 antibody:

For SLC22A2 (Transporter) Antibodies:

• Recommended dilution: 1:200 for Anti-SLC22A2 (OCT2) Antibody (#ACT-020)

• Validated positive controls: Mouse and rat kidney lysates

• Expected molecular weight: Varies based on species and post-translational modifications

• Blocking recommendation: Standard blocking buffer with 5% non-fat milk or BSA

For POU2F2 (Transcription Factor) Antibodies:

• Recommended dilution: 1:500-1:1000 for polyclonal antibody (10867-2-AP)

• Validated positive controls: NIH/3T3 cells, mouse ovary tissue

• Expected molecular weight: Calculated 44 kDa; Observed 55 kDa

• Sample preparation: Standard protein extraction protocols for nuclear proteins

For both antibody types, optimization may be required for different experimental systems, and titration of antibody concentrations is recommended to achieve optimal signal-to-noise ratios.

What are the best practices for OCT2 antibody use in immunohistochemistry of different tissue types?

Immunohistochemistry protocols should be tailored to the specific OCT2 protein and tissue type:

For SLC22A2 (Transporter) in Kidney Tissues:

• For FFPE mouse kidney sections: Use 1:100 dilution of Anti-SLC22A2 (OCT2) Antibody (#ACT-020)

• Antigen retrieval: Heat-induced epitope retrieval with citrate buffer at pH 6.0

• Detection system: Goat anti-rabbit-AlexaFluor-647 secondary antibody

• Expected pattern: OCT2 immunoreactivity in proximal tubules

• Control: Pre-incubation with SLC22A2 blocking peptide should suppress staining

For SLC22A2 (Transporter) in Brain Tissues:

• For perfusion-fixed frozen rat brain sections: Use 1:200 dilution of Anti-SLC22A2 (OCT2) Antibody (#ACT-020)

• Detection system: Goat anti-rabbit-AlexaFluor-488 secondary antibody

• Expected pattern: OCT2 immunoreactivity in neurons

• Control: Pre-incubation with SLC22A2 blocking peptide should suppress staining

For POU2F2 (Transcription Factor) in Lymphoid Tissues:

• For human lymphoma tissue: Use 1:20-1:200 dilution (optimization required)

• Antigen retrieval: Recommended with TE buffer at pH 9.0 or alternative with citrate buffer at pH 6.0

• Expected pattern: Nuclear staining in B-cells and B-cell derived lymphomas

• Positive controls: Germinal center B-cells, mantle B-cells, and plasma cells

How do I optimize immunoprecipitation protocols using OCT2 antibodies?

Immunoprecipitation with OCT2 antibodies requires careful optimization:

For POU2F2 (Transcription Factor):

• Recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Validated cell type: NIH/3T3 cells

• Protocol considerations:

  • Use gentle lysis buffers to preserve protein-protein interactions

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

  • Incubate antibody with lysate overnight at 4°C for optimal binding

  • Wash beads thoroughly (at least 3-5 times) to reduce background

  • Elute under denaturing conditions for subsequent SDS-PAGE analysis

For SLC22A2 (Transporter):

• No specific IP protocol was mentioned in the search results, but general IP principles would apply

• Consider using membrane protein-specific lysis buffers containing mild detergents

• May require optimization of detergent type and concentration to solubilize membrane proteins effectively without disrupting antibody binding

How can OCT2 antibodies be used in ChIP experiments to study transcription factor binding?

Chromatin immunoprecipitation (ChIP) is particularly relevant for POU2F2 as a transcription factor:

• The POU2F2 antibody (10867-2-AP) has been validated for ChIP applications

• Recommended protocol elements:

  • Use optimized cross-linking conditions (typically 1% formaldehyde for 10-15 minutes)

  • Ensure adequate chromatin shearing to 200-500 bp fragments

  • Use 2-5 μg of antibody per ChIP reaction

  • Include appropriate positive controls (genes known to be regulated by OCT2)

  • Include negative controls (IgG and gene desert regions)

  • Validate ChIP-qPCR primers for known OCT2 binding sites

  • Consider the octamer consensus sequence (ATGCAAAT) when designing primers

POU2F2 binds to the Ig gene octamer sites, making these regions excellent positive controls for ChIP experiments . This application is particularly relevant for studying B-cell differentiation and lymphoma research.

What are the considerations when using OCT2 antibodies in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence allows simultaneous detection of multiple proteins:

For SLC22A2 (Transporter):

• Compatible fluorophores: AlexaFluor-488 has been validated for brain tissue and AlexaFluor-647 for kidney tissue

• Nuclear counterstain: DAPI has been successfully used in combination

• Special considerations: Expression pattern is highly tissue-specific (proximal tubules in kidney, neurons in brain)

For POU2F2 (Transcription Factor):

• Consider panel design for B-cell research: Combine with other B-cell markers (CD20, CD79a) or other transcription factors

• Nuclear localization: Plan for proper nuclear counterstaining

• Primary antibody host considerations: The available antibodies include mouse monoclonal and rabbit polyclonal options, which should be considered when designing multiplexed panels to avoid cross-reactivity of secondary antibodies

General considerations for multiplexed studies:

• Carefully validate each antibody individually before combining

• Test for spectral overlap and implement appropriate controls

• Consider sequential staining protocols for antibodies raised in the same species

• Optimize each antibody's concentration in the multiplex setting

• Include appropriate blocking steps between sequential antibody applications

How do post-translational modifications affect OCT2 antibody binding and epitope recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition:

For SLC22A2 (Transporter):

• The Anti-SLC22A2 (OCT2) Antibody (#ACT-020) targets a peptide corresponding to amino acid residues 321-334 of mouse SLC22A2, located in the intracellular 3rd loop

• PTMs in this region could potentially affect antibody binding

• Phosphorylation sites in intracellular loops could be particularly relevant

For POU2F2 (Transcription Factor):

• Different antibodies target distinct regions:

  • Oct2-2136 targets amino acids 112-297

  • Other antibodies target regions including AA 11-100, AA 1-400, AA 277-389, and the N-terminus

• Transcription factors often undergo phosphorylation, acetylation, and SUMOylation

• These modifications may mask epitopes or alter protein conformation

• Differences between calculated MW (44 kDa) and observed MW (55 kDa) could be due to PTMs

Researchers should consider whether experimental conditions might alter the PTM status of the target protein, potentially affecting antibody recognition and experimental outcomes.

What controls should be included when using OCT2 antibodies for experimental validation?

Proper controls are essential for reliable interpretation of results:

For SLC22A2 (Transporter) Antibodies:

• Positive tissue controls: Mouse or rat kidney lysates for Western blot; kidney sections for IHC

• Negative controls: Non-expressing tissues or blocking peptide pre-incubation

• Peptide blocking control: Pre-incubation with SLC22A2 (OCT2) Blocking Peptide (BLP-CT020) should suppress specific staining

For POU2F2 (Transcription Factor) Antibodies:

• Positive controls: NIH/3T3 cells, mouse ovary tissue for Western blot; human lymphoma tissue for IHC

• Isotype controls: For monoclonal antibodies, include matching IgG isotype (e.g., IgG1 kappa for Oct2-2136)

• Cellular localization controls: POU2F2 should demonstrate nuclear localization

General Controls:

• Secondary antibody-only controls to assess background

• Loading controls for Western blot (e.g., β-actin, GAPDH)

• Titration of primary antibody to determine optimal concentration

• Multiple detection methods to confirm results (e.g., WB and IHC)

Including these controls provides crucial validation of antibody specificity and experimental reliability.

How do I address discrepancies in OCT2 detection between different experimental approaches?

When encountering conflicting results between different techniques:

• Confirm antibody specificity:

  • Verify that the same antibody is being used across experiments

  • Check epitope location relative to protein domains and potential splice variants

  • Confirm antibody performance with positive and negative controls

• Consider technical factors:

  • Fixation can mask epitopes in IHC/IF but not affect Western blot

  • Denaturation in Western blot may expose epitopes hidden in native conformation

  • For SLC22A2, membrane protein extraction methods significantly impact detection

  • For POU2F2, nuclear extraction efficiency may vary between protocols

• Sample-specific considerations:

  • Expression levels may vary between tissues/cell types

  • POU2F2 expression is highly regulated in B-cell differentiation

  • SLC22A2 shows tissue-specific expression patterns (kidney vs. brain)

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Combine antibody-based methods with non-antibody techniques (e.g., qPCR, mass spectrometry)

  • Consider knockout/knockdown validation

  • For POU2F2, perform functional assays (e.g., reporter gene assays)

How are OCT2 antibodies used to study drug transport mechanisms and nephrotoxicity?

SLC22A2 (OCT2) plays critical roles in drug transport and kidney function:

• Research applications:

  • Investigating drug-drug interactions at the transporter level

  • Studying mechanisms of nephrotoxicity for cationic drugs

  • Examining the impact of genetic polymorphisms on transporter function

  • Evaluating drug excretion mechanisms

• Methodological approaches:

  • Immunohistochemistry to localize OCT2 in proximal tubules

  • Western blot to quantify expression levels in kidney tissue

  • Co-localization studies with nephrotoxic compounds

  • Comparative analysis between healthy and diseased kidney tissues

Understanding SLC22A2 expression and localization provides insights into mechanisms of drug disposition and toxicity, particularly for organic cations and related compounds transported by this protein.

What is the significance of OCT2 antibodies in lymphoma research and classification?

POU2F2 (OCT2) serves as an important marker in lymphoma research:

• B-cell development and lymphomagenesis:

  • OCT2 expression varies across B-cell differentiation stages

  • High-level expression in germinal center B-cells, mantle B-cells, monocytoid B-cells, and plasma cells

• Diagnostic applications:

  • OCT2 expression is increased in multiple B-cell lymphoma types, including mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, plasmacytoma, Burkitt lymphoma, diffuse large cell lymphoma, diffuse large B-cell lymphoma, and Hodgkin lymphoma

  • Can aid in distinguishing B-cell from T-cell malignancies

• Research applications:

  • Studying transcriptional regulation in normal and malignant B-cells

  • Investigating OCT2's role in lymphomagenesis

  • Exploring potential as a therapeutic target

OCT2 antibodies provide valuable tools for lymphoma classification and for investigating the molecular mechanisms underlying B-cell malignancies.

How can researchers distinguish between the two different OCT2 proteins in their experimental systems?

Distinguishing between SLC22A2 and POU2F2 is crucial for experimental design and interpretation:

To ensure clarity in research:

• Always specify which OCT2 protein is being studied (SLC22A2 or POU2F2)

• Select antibodies specifically validated for your target protein

• Include functional assays appropriate to each protein's role

• Consider cellular localization studies (membrane vs. nuclear) to confirm identity

• Validate findings with gene-specific approaches (e.g., siRNA knockdown)

This careful distinction prevents confusion in experimental design, data interpretation, and research communication.