PAK2 (Ab-192) Antibody

What is PAK2 (Ab-192) Antibody and what epitope does it recognize?

PAK2 (Ab-192) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes p21-activated kinase 2 (PAK2). The antibody is generated using a synthesized non-phosphopeptide derived from human PAK2 around the phosphorylation site of serine 192 (T-K-S(p)-I-Y). It detects endogenous levels of total PAK2 protein regardless of phosphorylation status at this site .

What are the validated applications for PAK2 (Ab-192) Antibody?

The antibody has been validated for Western Blot (WB) and ELISA applications. For Western Blot applications, the recommended dilution range is 1:500-1:3000. The antibody has been specifically validated using HepG2 cell extracts treated with serum (20%, 15mins) .

What is the species reactivity profile of PAK2 (Ab-192) Antibody?

PAK2 (Ab-192) Antibody has been validated to react with human and mouse PAK2 proteins. This cross-species reactivity makes it particularly useful for comparative studies investigating conserved functions of PAK2 across these species .

What is the recommended storage protocol for maintaining antibody integrity?

Upon receipt, PAK2 (Ab-192) Antibody should be stored at -20°C or -80°C. For optimal preservation of activity, repeated freeze-thaw cycles should be avoided. The antibody is supplied in a formulation containing rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

How does PAK2 (Ab-192) Antibody compare with N-terminal and C-terminal specific PAK2 antibodies?

PAK2 (Ab-192) Antibody differs from other commercially available PAK2 antibodies in its epitope specificity. While PAK2 (Ab-192) targets an epitope around serine 192, other antibodies like P192 (NT) recognize epitopes near the N-terminus, and P191 (CT) recognize epitopes near the C-terminus of PAK2 . The choice between these antibodies should be determined by the specific research question. For example:

What methodological approaches can optimize Western blot results with PAK2 (Ab-192) Antibody?

To achieve optimal Western blot results with PAK2 (Ab-192) Antibody, consider the following methodological approach:

• Sample preparation: Lyse cells in a buffer containing protease and phosphatase inhibitors

• Protein loading: Load 20-40 μg of total protein per lane

• Gel separation: Use 10-12% SDS-PAGE gels for optimal resolution of PAK2 (58-62 kDa)

• Transfer conditions: Transfer to PVDF membrane at 100V for 60-90 minutes

• Blocking: Block with 5% non-fat dry milk in TBS-T for 1 hour at room temperature

• Primary antibody: Dilute PAK2 (Ab-192) Antibody 1:500-1:3000 in blocking buffer

• Incubation: Incubate overnight at 4°C with gentle rocking

• Washing: Wash 3-5 times with TBS-T, 5-10 minutes each

• Detection: Use HRP-conjugated secondary antibody and ECL detection system

How can researchers troubleshoot non-specific binding when using PAK2 (Ab-192) Antibody?

When encountering non-specific binding with PAK2 (Ab-192) Antibody, implement these methodological approaches:

• Increase antibody dilution (try 1:3000 instead of 1:500)

• Extend blocking time to 2 hours or use alternative blocking agents (BSA instead of milk)

• Increase wash frequency and duration between antibody incubations

• Pre-adsorb the antibody with cell/tissue lysate from a species different from your target

• Reduce primary antibody incubation time or temperature

• Include additional detergent (increase Tween-20 concentration in wash buffer to 0.1-0.2%)

• Use freshly prepared reagents and buffers

How can PAK2 (Ab-192) Antibody be utilized to investigate PAK2's dual role in cell survival and apoptosis?

PAK2 functions as a regulatory switch between cell survival and cell death signaling. To investigate this dual role using PAK2 (Ab-192) Antibody, implement the following experimental approaches:

• Compare full-length PAK2 (58-62 kDa) and the proapoptotic PAK-2p34 fragment (34 kDa) by Western blot after apoptotic stimuli

• Perform time-course experiments after treatment with apoptosis inducers like cisplatin

• Use PAK2 (Ab-192) Antibody in combination with caspase activity assays to correlate PAK2 cleavage with caspase activation

• Compare PAK2 expression patterns in cells with differential apoptotic responses

• Couple with subcellular fractionation to track PAK2 localization during apoptosis induction

Research findings demonstrate that full-length PAK2 stimulates cell survival and growth, while caspase-mediated cleavage generates the proapoptotic PAK-2p34 fragment involved in cell death responses. This dual functionality makes PAK2 a critical regulatory node in cell fate decisions .

How does PAK2 activity level correlate with cancer cell phenotypes, and how can this be studied?

PAK2 activity levels correlate with cancer progression, particularly in breast cancer models. PAK2 (Ab-192) Antibody can be employed to investigate these correlations through:

• Comparative analysis of PAK2 protein levels and activity across cancer cell lines with varying invasive potential

• In-gel kinase assays using myelin basic protein as substrate to measure PAK2 activity

• Correlation of PAK2 expression with anchorage-independent growth and resistance to apoptosis

• Conditional activation/inhibition studies to establish causality between PAK2 activity and malignant phenotypes

This data demonstrates that cancer cells with high PAK2 activity show reduced sensitivity to anticancer drug-induced apoptosis and lower levels of caspase activation of PAK2 .

What experimental approaches can be used to study PAK2's role in resistance to anticancer drugs?

PAK2 contributes to resistance to anticancer drugs by suppressing apoptotic responses. To investigate this role using PAK2 (Ab-192) Antibody:

• Compare PAK2 activity levels between drug-sensitive and drug-resistant cell lines

• Monitor changes in PAK2 expression and phosphorylation status during development of drug resistance

• Correlate PAK2 activity with caspase 3 activation and drug-induced apoptosis

• Implement conditional PAK2 activation/inhibition to determine causality

• Measure the ratio of full-length PAK2 to PAK-2p34 fragment as a potential biomarker of drug response

Research findings indicate that conditional activation of PAK2 suppresses activation of caspase 3, caspase activation of PAK2, and apoptosis in response to the anticancer drug cisplatin. This suggests a novel mechanism by which elevated PAK2 activity interrupts the apoptotic response and contributes to drug resistance in cancer cells .

How can PAK2 (Ab-192) Antibody be used to investigate PAK2's interaction with upstream regulators like CDC42 and RAC1?

PAK2 acts as a downstream effector of small GTPases CDC42 and RAC1. To study these interactions:

• Use PAK2 (Ab-192) Antibody in co-immunoprecipitation experiments followed by CDC42/RAC1 detection

• Perform pull-down assays with GST-PAK2 binding domain to measure active GTPases, followed by PAK2 detection

• Employ proximity ligation assays to visualize PAK2-GTPase interactions in situ

• Conduct FRET/BRET experiments combining the antibody with fluorescently tagged GTPases

• Implement cellular fractionation to track co-localization of PAK2 and GTPases in different cellular compartments

Research shows that activation of PAK2 by binding of active CDC42 and RAC1 results in a conformational change and subsequent autophosphorylation on several serine and/or threonine residues, leading to full activation of PAK2's kinase activity .

What considerations should be made when using PAK2 (Ab-192) Antibody for phosphorylation status analysis?

When studying PAK2 phosphorylation:

• PAK2 (Ab-192) Antibody recognizes total PAK2 around serine 192, regardless of phosphorylation status

• To specifically detect phosphorylated forms, combine with phospho-specific antibodies targeting key regulatory sites

• Use appropriate phosphatase inhibitors during sample preparation

• Include positive controls (serum-stimulated cells) that induce PAK2 phosphorylation

• Consider using Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated PAK2

• Implement 2D gel electrophoresis to resolve different phosphorylated forms

• Verify phosphorylation status with mass spectrometry for definitive identification

Research has shown that PAK2 activity migrates as two bands of approximately 58 and 62 kDa, with the 58 kDa band corresponding to highly active PAK2 despite containing lower protein levels than the 62 kDa band .

How might PAK2 (Ab-192) Antibody contribute to identification of novel therapeutic targets in cancer?

PAK2 (Ab-192) Antibody can facilitate the identification of novel therapeutic targets by:

• Screening for PAK2 interacting partners that mediate resistance to apoptosis

• Mapping the signaling networks downstream of PAK2 in cancer cells

• Identifying PAK2 substrates involved in cell survival pathways

• Evaluating PAK2 as a biomarker for predicting response to anticancer therapies

• Developing combination therapies targeting PAK2 and related pathways

The evidence that PAK2 is among 186 genes differentially expressed in breast cancer cells and strongly associated with the risk of metastasis and mortality underscores its potential as a therapeutic target .

What methodological approaches can be used to study the functional consequences of PAK2 phosphorylation at serine 192?

To study the specific role of serine 192 phosphorylation in PAK2 function:

• Generate site-specific phospho-mimetic (S192D/E) and phospho-deficient (S192A) PAK2 mutants

• Express these mutants in PAK2-depleted cells and analyze phenotypic consequences

• Use PAK2 (Ab-192) Antibody to confirm expression levels of mutant proteins

• Perform in vitro kinase assays to compare catalytic activities

• Identify kinases responsible for S192 phosphorylation using kinase inhibitors and siRNA approaches

• Map downstream signaling pathways affected by S192 phosphorylation status

How can PAK2 (Ab-192) Antibody be used in conjunction with imaging techniques to analyze PAK2 localization and dynamics?

To analyze PAK2 localization and dynamics:

• Use PAK2 (Ab-192) Antibody for immunofluorescence staining to visualize endogenous PAK2 distribution

• Combine with confocal microscopy to track PAK2 localization during cell cycle progression or apoptosis

• Implement live-cell imaging with fluorescently-tagged PAK2 and confirm results with antibody staining

• Use fluorescence recovery after photobleaching (FRAP) to study PAK2 mobility in different cellular compartments

• Employ super-resolution microscopy techniques to resolve PAK2 localization at the nanoscale level

• Perform correlative light and electron microscopy (CLEM) for ultrastructural localization studies

What controls should be included when using PAK2 (Ab-192) Antibody to ensure experimental validity?

To ensure robust and reproducible results:

• Positive control: Include serum-stimulated HepG2 cells, which have been validated for this antibody

• Negative control: Use PAK2 knockout/knockdown cells to confirm specificity

• Blocking peptide control: Pre-incubate antibody with immunizing peptide to verify specificity

• Loading control: Include detection of housekeeping proteins (β-actin, GAPDH) to normalize protein loading

• Secondary antibody control: Omit primary antibody to assess non-specific binding of secondary antibody

• Cross-reactivity control: Test the antibody against recombinant PAK1, PAK3, and PAK4 to confirm specificity within the PAK family

How can researchers standardize quantification of PAK2 activity across different experimental systems?

For standardized PAK2 activity quantification:

• Establish a reference cell line with well-characterized PAK2 expression and activity levels

• Create a standard curve using recombinant active PAK2 protein

• Implement in-gel kinase assays with myelin basic protein as substrate

• Normalize PAK2 activity to total PAK2 protein levels

• Use phospho-specific antibodies against key autophosphorylation sites as activity markers

• Include internal controls in each experiment to account for inter-assay variability

• Document detailed protocols for cell culture conditions, lysis methods, and activity measurements