phc2 Antibody

Introduction to PHC2 and PHC2 Antibodies

PHC2 (Polyhomeotic-like protein 2) is a nuclear protein that functions as a constituent of evolutionarily conserved protein complexes designated as class II PcG complexes . Also known as PH2, this protein plays a crucial role in anterior-posterior (A-P) specification through the transcriptional regulation of Hox cluster genes . PHC2 antibodies are immunological reagents specifically designed to detect and isolate the PHC2 protein in various experimental contexts, enabling researchers to investigate its functions, interactions, and cellular localization.

The significance of PHC2 in developmental processes has been highlighted through studies with PHC2-deficient mice, which display posterior transformations of the axial skeleton and premature senescence of mouse embryonic fibroblasts . These effects are associated with the derepression of Hox cluster genes and Cdkn2a genes, respectively, underscoring the importance of PHC2 in gene regulation .

Types of PHC2 Antibodies

PHC2 antibodies are available in various formats, each with specific characteristics and applications. They can be broadly categorized into monoclonal and polyclonal antibodies, with different conjugation options for specialized applications.

Monoclonal PHC2 Antibodies

Monoclonal antibodies against PHC2 offer high specificity and consistent binding properties. A notable example is the rabbit monoclonal antibody EPR27147-176 (ChIP Grade) produced by Abcam . This antibody is recombinantly produced (animal-free), providing several advantages:

• High batch-to-batch consistency and reproducibility

• Improved sensitivity and specificity

• Long-term security of supply

• Animal-free production

The same antibody is also available with Alexa Fluor® 647 conjugation (Ex: 652nm, Em: 668nm), enhancing its utility in fluorescence-based applications .

Polyclonal PHC2 Antibodies

Several polyclonal antibodies targeting PHC2 are commercially available, including:

• Proteintech's rabbit polyclonal antibody (12867-1-AP)

• Antibody Research Corporation's PHC2 Pab

• Merck Millipore's ABE1069

Polyclonal antibodies recognize multiple epitopes on the PHC2 protein, potentially increasing sensitivity but with potential variation between batches. The 12867-1-AP antibody, for instance, has been extensively validated for applications such as Western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence .

Applications of PHC2 Antibodies

PHC2 antibodies have been validated for numerous research applications, making them versatile tools in molecular and cellular biology research.

Western Blot (WB)

Western blotting is a common application for PHC2 antibodies, allowing for the detection and semi-quantification of PHC2 protein in cell or tissue lysates. The Proteintech antibody (12867-1-AP) has been validated for WB at dilutions of 1:500-1:1000 . Positive WB reactions have been detected in various cell lines including A375 and A549 cells . The observed molecular weight of PHC2 in Western blots is approximately 40 kDa, which differs from the calculated molecular weight of 91 kDa (858 amino acids) .

Immunohistochemistry (IHC)

PHC2 antibodies are effective for detecting the protein in tissue sections through immunohistochemistry. The recommended dilution for Proteintech's antibody in IHC applications is 1:50-1:500 . Positive IHC results have been observed in human pancreas cancer tissue and mouse kidney tissue . For optimal results, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative option .

Immunofluorescence (IF)/Immunocytochemistry (ICC)

Immunofluorescence and immunocytochemistry applications allow for the visualization of PHC2's subcellular localization. The Abcam monoclonal antibody has been validated for ICC/IF applications, with positive controls including SW480 and NIH/3T3 cells . The Alexa Fluor® 647-conjugated version (ab320782) is particularly useful for direct fluorescence detection in IF/ICC applications .

Other Applications

Beyond the applications mentioned above, PHC2 antibodies have been validated for:

• Immunoprecipitation (IP) with positive detection in HL-60 cells

• Flow cytometry (intracellular) with the Abcam monoclonal antibody

• ChIP-sequencing applications with the ChIP-grade monoclonal antibody from Abcam

• ELISA

Reactivity and Specificity of PHC2 Antibodies

The available PHC2 antibodies exhibit cross-reactivity with samples from multiple species:

It's worth noting that the Abcam monoclonal antibody (EPR27147-176) has been specifically identified as unsuitable for flow cytometry with intracellular human samples .

Monoclonal PHC2 Antibodies

The Abcam rabbit monoclonal antibody (EPR27147-176) is produced using a patented hybridoma-based technology (RabMAb®) . This antibody:

• Is generated against a recombinant fragment of human PHC2

• Has been validated with positive controls including SW480, Raji, NIH/3T3, NCCIT cells, and various tissue lysates

• Is provided in liquid form with preservatives and stabilizers

• Is purified using Protein A

The Alexa Fluor® 647-conjugated version maintains the same specificity while adding fluorescent properties for direct detection .

Polyclonal PHC2 Antibodies

The Proteintech polyclonal antibody (12867-1-AP) has the following characteristics:

• Host/Isotype: Rabbit/IgG

• Immunogen: PHC2 fusion protein Ag3507

• Observed molecular weight: 40 kDa

• Purification method: Antigen affinity purification

• Storage buffer: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

The Merck Millipore polyclonal antibody (ABE1069) is:

• Affinity purified

• Provided in a buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide

• Stable for 1 year when stored at 2-8°C

Developmental Biology Studies

Studies utilizing PHC2 antibodies have revealed crucial insights into developmental processes. Research on Phc2-deficient mice demonstrated posterior transformations of the axial skeleton, associated with derepression of Hox cluster genes . These findings highlight PHC2's role in anterior-posterior specification and embryonic development.

Cellular Localization and Interaction Studies

Immunofluorescence studies using PHC2 antibodies have provided valuable information about the protein's cellular localization and interactions. Co-immunofluorescence experiments have shown that PHC2 is concentrated inside CBX2 condensates in cells, with a moderate positive correlation of intensities between CBX2 and PHC2 condensates .

Research indicates that RING1B, BMI1, PHC1, PHC2, and H3K27me3 are all enriched within CBX2 condensates, suggesting a complex interaction network within the PRC1 complex .

Cell Cycle and Senescence Studies

PHC2 antibodies have facilitated investigations into cell cycle regulation and senescence. Studies with Phc2-deficient mice revealed premature senescence of mouse embryonic fibroblasts, which was associated with derepression of Cdkn2a genes . These findings suggest a role for PHC2 in cell cycle regulation and cellular aging processes.

Protocols and Recommended Dilutions for PHC2 Antibodies

For optimal results with PHC2 antibodies, specific protocols and dilutions are recommended based on the application and antibody source.

Recommended Dilutions for Proteintech 12867-1-AP

Note: It is recommended that each antibody should be titrated in specific testing systems to obtain optimal results, as results may be sample-dependent .

Future Perspectives in PHC2 Research

PHC2 antibodies continue to be essential tools for expanding our understanding of Polycomb group protein functions in development, gene regulation, and disease. Future research directions may include:

• Further characterization of PHC2's role in specific developmental contexts

• Investigation of PHC2's potential involvement in cancer and other diseases

• Deeper understanding of the molecular mechanisms underlying PHC2-mediated gene repression

• Examination of PHC2's interactions with other nuclear proteins and chromatin components

With the continued development and refinement of PHC2 antibodies, including recombinant monoclonal antibodies with improved consistency and specificity, researchers are well-equipped to address these questions and advance our understanding of this important epigenetic regulator.