P4H12 Antibody

What is the 4H12 monoclonal antibody and what target does it recognize?

The 4H12 monoclonal antibody is a murine antibody (IgG2a κ isotype) that specifically recognizes Myosin-9 (MYH9), also known as NMHCIIA. This antibody was originally generated using hybridoma technology against Faraz-ICR, a pancreatic acinar cell carcinoma cell line. The target antigen was identified through immunoprecipitation, Western blot, and mass spectrometry analysis, which revealed a protein with a molecular weight of approximately 250 kDa . The identification of MYH9 as the target antigen makes 4H12 particularly valuable for studying the role of this protein in pancreatic cancer biology and prognosis.

How is MYH9 expression distributed in pancreatic cancer tissues?

MYH9 expression varies significantly across pancreatic cancer tissues. Immunohistochemical staining using the 4H12 antibody revealed that in pancreatic ductal adenocarcinoma (PDAC) cases, approximately 42.8% expressed MYH9 with low intensity, while 47.8% and 9.5% of cases expressed MYH9 with moderate and strong intensities, respectively . Moreover, only about 28.6% of PDAC cases demonstrated both membranous and cytoplasmic staining, suggesting differential subcellular localization patterns. The acinar cell tumor from which the Faraz-ICR cell line was derived showed high cytoplasmic expression of MYH9, indicating potential significance in acinar cell carcinoma biology .

What experimental controls are recommended when using 4H12 antibody in flow cytometry?

When using 4H12 antibody (or any antibody) in flow cytometry experiments, researchers should implement several controls to ensure result validity:

• Unstained cells control: To account for autofluorescence from endogenous fluorophores that may increase false positives .

• Negative cells control: Cell populations not expressing MYH9 should be used to confirm the target specificity of the 4H12 antibody .

• Isotype control: An IgG2a κ antibody with no known specificity or generated against an antigen not present in the cell population should be used to assess background staining due to Fc receptor binding .

• Secondary antibody control: For indirect staining protocols, cells treated with only the labeled secondary antibody should be prepared to address non-specific binding of the secondary antibody .

Additionally, researchers should block non-specific binding sites using 10% normal serum from the same host species as the labeled secondary antibody (but not from the same host species as the primary antibody) .

How does the subcellular localization of MYH9 differ across cancer cell lines when detected with 4H12 antibody?

Flow cytometry analysis using 4H12 antibody has revealed interesting patterns of MYH9 subcellular localization across different cancer cell lines:

This differential expression pattern is significant because previous studies had not demonstrated the expression of MYH9 at the surface of cancer cells using flow cytometry. The membranous expression of MYH9 may facilitate viral infections by acting as a receptor for sialylated RNA viruses . These findings suggest that 4H12 antibody could be a valuable tool for investigating the role of MYH9 in different cancer types and its potential as a diagnostic or therapeutic target.

What inhibitory effects does 4H12 antibody demonstrate on pancreatic cancer cell proliferation?

The 4H12 monoclonal antibody has shown significant anti-proliferative effects on Faraz-ICR pancreatic acinar cell carcinoma cells. Experimental data demonstrates dose-dependent inhibition of cell proliferation across a concentration range of 0.75 to 12.5 μg/ml (p < 0.0001 and p < 0.002) .

The inhibitory potency of 4H12 antibody can be quantified by its IC50 values:

• After 24 hours of treatment: 12.09 ± 4.19 μg/ml

• After 48 hours of treatment: 7.74 ± 4.28 μg/ml

This decrease in IC50 value with longer exposure indicates increasing efficacy over time, suggesting that the 4H12 antibody could potentially be developed as a therapeutic agent targeting MYH9-expressing pancreatic cancer cells . These findings highlight the importance of investigating the mechanisms through which 4H12 antibody inhibits cancer cell proliferation.

How should researchers optimize flow cytometry protocols when targeting MYH9 with 4H12 antibody?

When targeting MYH9 with 4H12 antibody using flow cytometry, researchers should optimize their protocols based on several considerations:

• Cellular localization: Since MYH9 shows both surface and intracellular expression in some cell lines, researchers must decide whether to perform surface staining only, intracellular staining only, or both. For comprehensive analysis, both approaches may be necessary .

• Fixation and permeabilization: For intracellular detection of MYH9, cells must be fixed to preserve cellular contents when the membrane is compromised. The fixation and permeabilization method should be optimized based on the epitope recognized by 4H12 antibody .

• Cell preparation:

  • Ensure cell viability >90% before staining, as dead cells give high background scatter and may show false positive staining

  • Use appropriate cell concentrations (10^5 to 10^6 cells) to avoid clogging the flow cell

  • Consider starting with higher cell numbers (10^7 cells/tube) if multiple washing steps are involved

  • Perform all steps on ice and use PBS with 0.1% sodium azide to prevent internalization of membrane antigens

• Antibody validation: Always use flow cytometry-validated antibodies when possible, as antibodies successfully tested for Western blotting or immunohistochemistry may not be suitable for flow cytometry .

What control cell lines should be used when studying MYH9 expression with 4H12 antibody?

When studying MYH9 expression using the 4H12 antibody, selection of appropriate control cell lines is crucial for experimental validity:

• Positive controls: Based on the available data, Faraz-ICR and MIA-PaCa2 pancreatic cancer cell lines show high expression levels of MYH9 (both surface and intracellular), making them excellent positive controls . Additionally, SW1116 colorectal cancer cells demonstrate high surface expression (95.8%) and could serve as positive controls for membrane-localized MYH9 .

• Negative controls: The PaTu 8902 pancreatic cancer cell line showed negative surface expression of MYH9, making it suitable as a negative control for surface staining experiments . Similarly, MDA-MB-231 and MCF-7 breast cancer cell lines showed negative surface expression and could be used as negative controls for membrane-localized MYH9 .

• Variable expression controls: SKOV3 ovarian cancer cells with intermediate surface expression (28.5%) and high intracellular expression (79.5%) could serve as variable expression controls .

For comprehensive study design, researchers should include:

• Cell lines from the same tissue of interest (e.g., pancreatic cell lines)

• Cell lines with known differential expression

• Non-cancerous control cells where appropriate

Before initiating experiments, researchers should verify MYH9 expression in their chosen cell lines through literature review or resources like The Human Protein Atlas .

How can researchers troubleshoot non-specific binding when using 4H12 antibody in flow cytometry?

Non-specific binding is a common challenge when using antibodies like 4H12 in flow cytometry. Researchers can address this issue through several approaches:

• Blocking optimization:

  • Use 10% normal serum from the same host species as the labeled secondary antibody

  • Ensure the blocking serum is NOT from the same host species as the primary antibody to avoid non-specific signals

  • Consider including 1% BSA in blocking and washing buffers to reduce non-specific binding

• Titration of antibody concentration:

  • Perform a titration series to determine the optimal concentration of 4H12 antibody that provides the best signal-to-noise ratio

  • Test different concentrations ranging from 0.1-10 μg/ml to identify the minimum concentration giving maximum specific signal with minimal background

• Pre-adsorption controls:

  • Pre-incubate 4H12 antibody with purified MYH9 protein before cell staining

  • If the staining is specific, pre-adsorption should significantly reduce or eliminate the signal

• Fc receptor blocking:

  • Use specific Fc receptor blocking reagents when working with cells known to express high levels of Fc receptors

  • This is particularly important for immune cells or cells derived from hematopoietic lineages

What methodological approaches should be used to confirm the specificity of 4H12 antibody for MYH9?

To confirm the specificity of 4H12 antibody for MYH9, researchers should employ multiple orthogonal methods:

• Immunoprecipitation and mass spectrometry:

  • Immunoprecipitate the target protein using 4H12 antibody

  • Perform SDS-PAGE and excise the band of interest

  • Analyze by LC-MS/MS to confirm identity as MYH9 (as was done in the original characterization)

• Western blot analysis:

  • Perform Western blotting using 4H12 antibody on various cell lines

  • Confirm the presence of a single band at approximately 250 kDa (the molecular weight of MYH9)

  • Compare with a commercial anti-MYH9 antibody as reference

• RNA interference validation:

  • Perform siRNA or shRNA knockdown of MYH9 in positive cell lines

  • Demonstrate reduced binding of 4H12 antibody proportional to knockdown efficiency

  • Include non-targeting siRNA controls to confirm specificity

• Immunofluorescence co-localization:

  • Perform co-staining with 4H12 antibody and a validated commercial anti-MYH9 antibody

  • Demonstrate co-localization using confocal microscopy

  • Include appropriate controls to rule out cross-reactivity

How can 4H12 antibody be used to investigate the role of MYH9 in pancreatic cancer prognosis?

The 4H12 antibody offers several approaches to investigate MYH9's role in pancreatic cancer prognosis:

• Tissue microarray analysis:

  • Use 4H12 antibody for immunohistochemical staining of pancreatic cancer tissue microarrays

  • Quantify MYH9 expression levels and correlate with patient survival data

  • Analyze associations between expression patterns (cytoplasmic vs. membranous) and clinical outcomes

  • The initial findings showing variable expression intensities (42.8% low, 47.8% moderate, 9.5% high) in PDAC cases suggest potential prognostic value

• Functional studies in patient-derived xenografts:

  • Treat patient-derived pancreatic cancer xenografts with 4H12 antibody

  • Monitor tumor growth, metastasis, and response to standard therapies

  • Correlate treatment efficacy with MYH9 expression levels

• Liquid biopsy applications:

  • Investigate whether 4H12 antibody can detect circulating tumor cells (CTCs) expressing MYH9

  • Correlate CTC counts and MYH9 expression with disease progression and treatment response

• Multi-marker prognostic panels:

  • Integrate MYH9 detection using 4H12 antibody with other established prognostic markers

  • Develop and validate a composite scoring system for improved prognostic accuracy

The anti-proliferative effects observed with 4H12 antibody treatment (IC50 values of 12.09 ± 4.19 μg/ml at 24h and 7.74 ± 4.28 μg/ml at 48h) further suggest its potential value in stratifying patients who might benefit from MYH9-targeted therapies .

What mechanisms might explain the inhibitory effect of 4H12 antibody on pancreatic cancer cell proliferation?

Several potential mechanisms could explain the observed inhibitory effect of 4H12 antibody on pancreatic cancer cell proliferation:

• Disruption of cytoskeletal functions:

  • MYH9 is a key component of the non-muscle myosin II complex involved in cell migration, adhesion, and cytokinesis

  • 4H12 antibody binding may interfere with these essential cellular processes

  • Impaired cytokinesis could lead to mitotic catastrophe and cell death

• Interference with signal transduction:

  • MYH9 has been implicated in several signaling pathways, including MAPK and Rho GTPase signaling

  • 4H12 antibody binding may disrupt protein-protein interactions necessary for proper signal transduction

  • This could inhibit pro-proliferative and pro-survival signaling cascades

• Induction of antibody-dependent cellular cytotoxicity (ADCC):

  • As an IgG2a isotype, 4H12 may potentially induce ADCC when binding to surface-expressed MYH9

  • This would trigger immune cell-mediated killing of antibody-coated cancer cells

  • This mechanism would be particularly relevant in in vivo settings

• Disruption of membranous MYH9 functions:

  • The discovery of surface expression of MYH9 in certain cancer cell lines suggests it may have previously unrecognized membrane-associated functions

  • 4H12 antibody may interfere with these functions, potentially including interactions with the extracellular matrix or other membrane proteins

Detailed mechanistic studies combining 4H12 antibody treatment with pathway inhibitors, cytoskeletal disruptors, and genetic approaches would help elucidate the precise mechanism underlying its anti-proliferative effects.

What is the optimal protocol for isotyping and purification of 4H12 antibody from hybridoma supernatants?

For optimal isotyping and purification of 4H12 antibody from hybridoma supernatants, researchers should follow this protocol:

• Hybridoma culture optimization:

  • Culture hybridoma cells in appropriate medium (typically RPMI-1640 with 10% FBS)

  • When cells reach optimal density, switch to serum-free medium for antibody production

  • Harvest supernatant when cell viability begins to decline

• Isotyping procedure:

  • Perform initial isotyping using commercial kits such as Pierce Rapid Antibody Isotyping Kits for mouse antibodies (as referenced in search result )

  • Confirm isotype results using a secondary method such as ELISA with isotype-specific secondary antibodies

  • For 4H12, expect IgG2a κ isotype confirmation

• Purification protocol:

  • For IgG2a antibodies like 4H12, Protein A or Protein G affinity chromatography is recommended

  • Condition the column with binding buffer (typically phosphate buffer, pH 7.4)

  • Apply filtered hybridoma supernatant to the column

  • Wash extensively to remove non-specific proteins

  • Elute bound antibody with low pH buffer (typically 0.1M glycine, pH 2.5-3.0)

  • Immediately neutralize eluted fractions with 1M Tris, pH 8.0

  • Dialyze against PBS or desired storage buffer

• Quality control:

  • Confirm purity by SDS-PAGE (expect heavy chain at ~50 kDa and light chain at ~25 kDa)

  • Measure concentration by absorbance at 280 nm (extinction coefficient for mouse IgG ≈ 1.4)

  • Verify activity using flow cytometry with Faraz-ICR cells as positive controls

  • Aliquot and store at -20°C or -80°C for long-term storage

How should researchers design experiments to compare intracellular versus surface expression of MYH9 using 4H12 antibody?

Designing experiments to compare intracellular versus surface expression of MYH9 using 4H12 antibody requires careful protocol optimization:

• Experimental groups setup:

  • Group A: Surface staining only

  • Group B: Intracellular staining only

  • Group C: Combined surface and intracellular staining

  • Group D: Appropriate controls (unstained, isotype, secondary antibody)

• Surface staining protocol:

  • Use unfixed, live cells maintained on ice

  • Incubate with 4H12 antibody in PBS containing 1% BSA and 0.1% sodium azide

  • Wash and incubate with fluorochrome-conjugated secondary antibody

  • Wash and analyze immediately without fixation

• Intracellular staining protocol:

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100

  • Block with appropriate serum

  • Incubate with 4H12 antibody in permeabilization buffer

  • Wash and incubate with fluorochrome-conjugated secondary antibody

  • Wash and analyze

• Combined sequential staining:

  • First perform surface staining with 4H12 antibody and a specific fluorochrome (e.g., FITC)

  • Wash thoroughly and fix cells

  • Permeabilize and perform intracellular staining with 4H12 and a different fluorochrome (e.g., PE)

  • This approach allows simultaneous visualization of surface and intracellular pools

• Data analysis strategies:

  • Calculate the ratio of surface to intracellular expression

  • Generate scatter plots comparing surface versus intracellular staining intensity

  • Consider using imaging flow cytometry to visualize subcellular localization patterns

This experimental design would allow researchers to quantitatively compare the relative distribution of MYH9 between membrane and cytoplasmic compartments across different cell types, potentially providing insights into its diverse cellular functions.