PLEKHA1 Antibody

What is PLEKHA1 and what is its significance in research?

PLEKHA1, also known as TAPP1 (tandem PH domain containing protein-1), is a protein containing pleckstrin homology (PH) domains that are commonly found in eukaryotic signaling proteins. These domains possess multiple functions including the ability to bind inositol phosphates and various proteins . The protein has a calculated molecular weight of 46 kDa, which matches its observed molecular weight in experimental conditions . PLEKHA1 is encoded by the gene with ID 59338 in NCBI databases .

The significance of PLEKHA1 in research lies in its involvement in cellular signaling pathways, which makes it relevant for studies in normal cellular processes and various disease states. Research on PLEKHA1 contributes to our understanding of phosphoinositide-mediated signaling and its implications in cellular functions and pathologies.

PLEKHA1 antibodies have demonstrated reactivity with various human samples and cell lines. Based on the search results, the following sample types have shown positive reactivity:

Tissues:

• Human placenta tissue

• Human colon tissue

• Human pancreas cancer tissue

• Human breast cancer tissue

• Human invasive urothelial carcinoma of the bladder

• Human lung cancer tissue

Cell Lines:

• Jurkat cells

• HeLa cells

• A431 cells

While most validation has been performed with human samples, there is cited reactivity with mouse samples as well , suggesting cross-reactivity potential across species though this would require specific validation for research purposes.

What are the optimal dilution ratios for different PLEKHA1 antibody applications?

Determining the optimal dilution for PLEKHA1 antibodies is crucial for experimental success. Based on the search results, the following dilutions are recommended for various applications:

It is important to note that these are general recommendations, and titration within these ranges is advised for each specific experimental system to obtain optimal results . Antibody performance can be sample-dependent, so validation with appropriate controls is essential for each new experimental context.

What antigen retrieval methods work best for PLEKHA1 IHC staining?

The choice of antigen retrieval method significantly impacts the quality of PLEKHA1 immunohistochemical staining. Based on the search results, the following antigen retrieval approaches have been successfully employed:

For paraffin-embedded tissue sections:

• Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended for optimal results

• Alternatively, antigen retrieval with TE buffer (pH 9.0) has been suggested

• Citrate buffer (pH 6.0) can also be used as an alternative approach

For immunocytochemistry applications:

• Enzyme antigen retrieval has been successfully applied using specialized reagents (e.g., AR0022) with an incubation time of 15 minutes

These different methods provide flexibility depending on the specific tissue type, fixation conditions, and laboratory preferences. The consistent recommendation for alkaline pH buffers (pH 8.0-9.0) suggests this provides better epitope accessibility for PLEKHA1 detection in fixed tissues.

How should PLEKHA1 antibodies be stored to maintain optimal performance?

Proper storage of PLEKHA1 antibodies is essential for maintaining their activity and specificity over time. According to the search results, the following storage conditions are recommended:

• Storage temperature: -20°C is the standard recommendation

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability: When stored properly, the antibodies are reported to be stable for one year after shipment

• Aliquoting: For small volume antibodies (e.g., 20μl sizes), aliquoting is specified as unnecessary for -20°C storage

• Some formulations may contain 0.1% BSA which contributes to antibody stability

To preserve antibody activity, it's advisable to minimize freeze-thaw cycles and handle the antibody at appropriate temperatures during experimental procedures. Following these storage recommendations will help ensure consistent experimental results over the antibody's shelf life.

What is the specificity of PLEKHA1 antibodies across different cellular contexts?

The specificity of PLEKHA1 antibodies varies across different cellular contexts and tissues, which is an important consideration for research applications. The search results reveal several important aspects of PLEKHA1 antibody specificity:

In normal tissues:

• PLEKHA1 antibodies show specific staining in human placenta tissue

• Colon tissue demonstrates detectable PLEKHA1 expression

In cancer tissues:

• Breast cancer tissues show PLEKHA1 expression patterns that can be detected by immunohistochemistry

• Lung cancer tissue samples demonstrate detectable PLEKHA1 levels

• Pancreatic cancer tissue shows reactivity

• Invasive urothelial carcinoma of the bladder with squamous differentiation exhibits PLEKHA1 expression

In cell lines:

• Jurkat cells (T lymphocyte cells) consistently show PLEKHA1 expression in western blot analyses

• HeLa cells demonstrate detectable PLEKHA1 by immunofluorescence

• A431 cells (epidermoid carcinoma cells) show PLEKHA1 expression suitable for immunofluorescence and flow cytometry applications

These patterns suggest PLEKHA1 may have context-dependent expression profiles that researchers should consider when designing experiments to study this protein in different biological systems or disease states.

What controls should be used when working with PLEKHA1 antibodies?

Incorporating appropriate controls is crucial for validating results obtained with PLEKHA1 antibodies. Based on the search results and standard research practices, the following controls are recommended:

Positive Controls:

• Jurkat cell lysates have been consistently demonstrated to express PLEKHA1 and serve as reliable positive controls for western blot

• Human placenta tissue serves as a positive control for IHC and IF applications

• HeLa and A431 cells are suitable positive controls for IF/ICC experiments

Negative Controls:

• Isotype control antibodies (e.g., rabbit IgG at equivalent concentrations) should be used to identify non-specific binding, particularly in flow cytometry applications

• Unlabelled samples serve as baselines for autofluorescence in flow cytometry

• Secondary antibody-only controls help identify background signal from non-specific secondary antibody binding

Loading Controls:

• For western blot applications, standard loading controls such as GAPDH, β-actin, or α-tubulin should be included to normalize protein loading

• Counterstaining with DAPI for nuclei identification in IF/ICC experiments enhances interpretation of subcellular localization

These controls help distinguish specific PLEKHA1 detection from technical artifacts and enable accurate interpretation of experimental results.

How can I troubleshoot weak or non-specific PLEKHA1 antibody signals?

Troubleshooting antibody performance issues requires systematic analysis of experimental conditions. Based on the search results and standard immunodetection practices, the following strategies can help address weak or non-specific signals when working with PLEKHA1 antibodies:

For Weak Signals:

• Antibody concentration: Adjust the dilution within the recommended range (1:50-1:500 for IHC/IF or 1:500-1:1000 for WB)

• Antigen retrieval: Optimize antigen retrieval methods by testing alternatives (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Incubation conditions: Extend primary antibody incubation time (e.g., overnight at 4°C as used in validation studies)

• Detection system: Use a more sensitive detection method or signal amplification system

• Sample preparation: Ensure proper protein extraction and denaturation for western blot applications

For Non-specific Signals:

• Blocking: Increase blocking time or concentration (e.g., 10% goat serum as used in validation protocols)

• Washing: Implement more stringent washing steps (e.g., TBS-0.1% Tween, 3 times for 5 minutes each as used in WB protocols)

• Antibody specificity: Consider using a different antibody that targets a different epitope of PLEKHA1

• Secondary antibody: Test alternative secondary antibodies or reduce their concentration to minimize background

• Sample-specific factors: Address autofluorescence or endogenous peroxidase activity in tissue samples

When optimizing conditions, it's advisable to change one parameter at a time and include appropriate controls to isolate the source of the problem.

What is the expected molecular weight for PLEKHA1 in western blots?

The expected molecular weight for PLEKHA1 in western blot applications is 46 kDa, which is consistent across multiple antibody sources and validation studies . This observation aligns with the calculated molecular weight based on the amino acid sequence of the protein .

When performing western blot analysis using PLEKHA1 antibodies, researchers should expect to detect a specific band at approximately 46 kDa, as confirmed in validation studies using human Jurkat cell lysates . The consistency between the calculated and observed molecular weights suggests minimal post-translational modifications that would significantly alter the protein's migration pattern in SDS-PAGE.

It's worth noting that the complete amino acid sequence of human PLEKHA1 (NP_001001974.1) includes 404 amino acids , and antibodies may be generated against different regions of this sequence. For example, some antibodies target the full-length protein (AA 1-404) , while others may target specific domains or regions, which could potentially affect the recognition pattern in certain experimental conditions.

What is the subcellular localization pattern for PLEKHA1?

Understanding the subcellular localization of PLEKHA1 provides insights into its potential functions. Based on the immunofluorescence data from the search results, PLEKHA1 demonstrates specific localization patterns that researchers should consider when interpreting their results:

In cell lines:

• Immunofluorescence studies in A431 cells revealed detectable PLEKHA1 expression with distinct localization patterns

• HeLa cells show positive immunofluorescence staining, indicating expression in this cell type

In tissue sections:

• Immunofluorescence analysis of human lung cancer tissue showed PLEKHA1 localization patterns that could be visualized using Cy3-conjugated secondary antibodies and counterstained with DAPI for nuclear visualization

When conducting immunofluorescence experiments to study PLEKHA1 localization, researchers should consider:

• Using appropriate counterstains to identify cellular compartments

• Co-staining with markers of specific organelles or structures to precisely determine localization

• Comparing localization patterns across different cell types or under different stimulation conditions

How do different tissue types compare in PLEKHA1 expression patterns?

PLEKHA1 expression varies across different tissue types, and understanding these patterns is valuable for research in both normal physiology and disease states. Based on the immunohistochemistry data from the search results, the following comparative patterns emerge:

Normal Tissues:

• Human placenta tissue shows detectable PLEKHA1 expression and serves as a positive control in validation studies

• Human colon tissue demonstrates PLEKHA1 expression that can be detected by IHC

Cancer Tissues:

• Human breast cancer tissue shows specific PLEKHA1 staining patterns

• Human pancreatic cancer tissue exhibits detectable PLEKHA1 expression

• Human lung cancer tissue demonstrates PLEKHA1 expression patterns

• Invasive urothelial carcinoma of the bladder with squamous differentiation shows PLEKHA1 expression

These observations suggest that PLEKHA1 is expressed in multiple tissue types, with potentially altered expression patterns in malignant states. The presence of PLEKHA1 across diverse tissue types indicates it may have broad physiological relevance rather than highly tissue-restricted functions.

For researchers studying PLEKHA1 in specific disease contexts:

• Consider comparing expression levels between normal and pathological tissues of the same origin

• Examine whether subcellular localization changes in disease states

• Investigate potential correlations between PLEKHA1 expression levels and clinical parameters or outcomes