ATP1A1 Antibody

What experimental applications are ATP1A1 antibodies commonly used for?

ATP1A1 antibodies have been validated for multiple experimental applications, with varying protocols and optimization requirements:

• Western Blotting (WB): Most ATP1A1 antibodies demonstrate strong performance in WB applications at dilutions ranging from 1:5000-1:20000. Note that for optimal detection with some antibodies, samples should be heated only to 37°C rather than boiling .

• Immunohistochemistry (IHC): Antibodies typically perform well at dilutions between 1:1000-1:4000, with both paraffin-embedded and frozen section compatibility .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): These applications generally require dilutions of 1:400-1:1600 .

• Flow Cytometry: For intracellular detection, approximately 0.40 μg per 10^6 cells in a 100 μl suspension is typically recommended .

• Immunoprecipitation (IP) and Co-Immunoprecipitation (CoIP): Between 0.5-4.0 μg of antibody is typically used for 1.0-3.0 mg of total protein lysate .

It's critical to note that titration is necessary in each experimental system to obtain optimal results, as sample-dependent factors can influence antibody performance .

What tissue reactivity can researchers expect from ATP1A1 antibodies?

Most ATP1A1 antibodies demonstrate reactivity against human, mouse, and rat samples . Some antibodies have also shown reactivity with samples from:

• Porcine tissues

• Canine tissues

• Bovine specimens

• Sheep tissues

• Chick models

• Rabbit tissues

• Horse tissues

When working with non-validated species, cross-reactivity testing should be performed before proceeding with full-scale experiments. Sequence analysis reveals that the human ATP1A1 differs from the mouse sequence by three amino acids and from the rat sequence by four amino acids in certain immunogenic regions .

How should researchers optimize sample preparation for ATP1A1 detection in Western blotting?

Sample preparation is critical for successful ATP1A1 detection:

• Heat Treatment: Unlike many proteins, ATP1A1 detection often requires non-standard heating protocols. Multiple sources specifically note that samples should not be boiled but instead heated only to 37°C prior to loading on SDS-PAGE gels .

• Protein Extraction: For membrane proteins like ATP1A1, extraction buffers containing mild detergents (such as 0.5-1% Triton X-100 or CHAPS) are generally more effective than harsher ionic detergents.

• Loading Control Selection: When performing quantitative analysis of ATP1A1 expression, researchers should select loading controls that have similar subcellular localization (other membrane proteins) rather than cytosolic proteins to ensure accurate normalization .

• Sample Source Optimization: Western blot analysis has been successfully demonstrated with:

  • HEK-293 cells

  • Mouse brain tissue

  • Mouse heart tissue

  • Rat heart tissue

  • Human Colo-205 colon adenocarcinoma cells

What controls should be implemented when studying ATP1A1 in cancer tissues?

When investigating ATP1A1 expression in cancer tissues, several controls are essential:

• Paired Normal-Tumor Samples: Always include matched normal tissue as a baseline comparison. Studies have shown differential expression of ATP1A1 across tumor grades, particularly in gliomas .

• Blocking Peptide Controls: Use blocking peptides specific to the antibody being employed to confirm specificity. Multiple commercial suppliers offer these peptides upon request .

• Grade-Specific Controls: For gliomas specifically, include tissues representing different WHO grades (I-IV) as ATP1A1 expression has been shown to correlate with tumor grade .

• Positive Control Tissues: Known high-expressing tissues that have been validated include:

  • Human ovarian tumor tissue

  • Human cervical cancer tissue

  • Human colon cancer tissue

  • Human liver cancer tissue

  • Human endometrioid adenocarcinoma tissue

How is ATP1A1 involved in cancer biology, particularly glioblastoma?

ATP1A1 demonstrates significant involvement in cancer biology, particularly in glioblastoma multiforme (GBM):

• Expression Correlation with Tumor Grade: Immunohistochemical analyses of glioma tissue arrays revealed that ATP1A1 expression increases progressively with tumor grade. High-grade gliomas (WHO grade III astrocytomas and GBMs) show significantly higher expression compared to low-grade gliomas (grade I and II astrocytomas) and normal brain tissue .

• Quantitative Relationship: The percentage of cells positively reacting with ATP1A1 antibody showed a direct correlation with the grade of GBM .

• Glioma Stem Cells (GSCs): ATP1A1 is remarkably overexpressed in GSCs compared to differentiated GBM cells in five out of seven GSC lines examined. These GSCs also expressed stemness markers nestin and SOX2 .

• Functional Role: Knockdown of ATP1A1 in GSCs using shRNA resulted in significantly decreased proliferation and survival at 48h and 72h after transfection, with sh-ATP1A1-1 showing the strongest effect. This suggests ATP1A1 functions as an oncogene in GSC models .

• Signaling Mechanism: ATP1A1 appears to interact with Src and affect the activation of ERK1/2 and AKT pathways, potentially explaining its role in promoting GSC proliferation and growth .

What role does ATP1A1 play in viral infection mechanisms?

ATP1A1 has been identified as a critical host factor for viral entry, particularly for respiratory syncytial virus (RSV):

• Essential for Viral Entry: ATP1A1 was identified among genes with the strongest effect on RSV-GFP infection in a screening study. Knockdown confirmation demonstrated that reducing ATP1A1 significantly impaired viral infection .

• Knockdown Efficiency: Using siRNAs targeting ATP1A1, researchers achieved reduction of ATP1A1 mRNA to below 5% compared to negative control, resulting in protein expression reduction to approximately 35-39% .

• Cellular Distribution During Infection: In uninfected cells, ATP1A1 is homogeneously distributed on the plasma membrane. Following RSV infection, ATP1A1 forms distinct clusters as early as 15 minutes post-infection, becoming more prominent and numerous over time .

• Co-localization with Viral Proteins: Some ATP1A1 clusters partially co-localize with RSV F protein during early infection stages, suggesting direct involvement in the viral entry process .

• Macropinocytic Entry Mechanism: ATP1A1 appears to be required specifically for macropinocytic entry of RSV in human respiratory epithelial cells, representing a critical host-pathogen interaction .

How can researchers address inconsistent ATP1A1 antibody staining results?

When facing inconsistent ATP1A1 staining, consider these methodological adjustments:

• Antigen Retrieval Optimization:

  • For paraffin-embedded tissues, use TE buffer at pH 9.0 as primary choice

  • Alternative: citrate buffer at pH 6.0

  • Heat-mediated retrieval has shown superior results compared to enzymatic methods for ATP1A1

• Blocking Protocol Adjustment:

  • 10% goat serum has been validated in multiple studies

  • Incubation for 1-2 hours at room temperature before primary antibody application

• Antibody Incubation Conditions:

  • Overnight incubation at 4°C with primary antibody (typically 2 μg/ml)

  • Secondary antibody incubation for 30 minutes at 37°C

• Tissue-Specific Considerations:

  • For brain tissue: immersion-fixed, free-floating frozen sections show excellent results

  • For kidney tissue: ATP1A1 staining is predominantly in collecting ducts and thick portions of the Henle loop in renal medulla

• Signal Amplification Systems:

  • HRP-conjugated detection systems with DAB as chromogen provide consistent results

  • For fluorescent detection, particularly in hippocampal tissue, combine with DAPI for nuclear visualization

What factors should be considered when selecting an ATP1A1 antibody for specific experimental systems?

Selection of the appropriate ATP1A1 antibody requires consideration of several factors:

• Epitope Location: Different antibodies target different regions of ATP1A1:

  • N-terminal antibodies (residues 7-20 in rat ATP1A1)

  • Mid-region antibodies

  • C-terminal antibodies

  • Phosphorylation-specific antibodies (pTyr10, pSer16)

• Species Cross-Reactivity Requirements: If working with non-human models, verify cross-reactivity. Most antibodies work with human, mouse, and rat samples, but validation in other species varies considerably .

• Application Compatibility:

  • For multi-application studies, select antibodies validated across all required techniques

  • For specialized applications (e.g., CoIP), choose antibodies specifically validated for that purpose

• Clonality Considerations:

  • Polyclonal antibodies often provide stronger signals but may have more background

  • Monoclonal antibodies typically offer higher specificity but potentially lower sensitivity

  • Current validated options include both rabbit polyclonal and mouse monoclonal antibodies

• Protein Modification Detection:

  • For studying phosphorylation states, specific antibodies targeting pTyr10 or pSer16 are available

  • These modified forms may reveal distinct functional states of ATP1A1

How should researchers quantitatively analyze ATP1A1 expression changes?

For reliable quantitative analysis of ATP1A1 expression:

• Western Blot Quantification:

  • Use housekeeping proteins of similar molecular weight range (90-130 kDa)

  • Perform densitometric analysis using software like ImageJ or specialized image analysis platforms

  • Report relative expression normalized to controls using at least three biological replicates

• IHC Scoring Systems:

  • For tissue arrays, quantify percentage of cells positively reacting with ATP1A1 antibody

  • In glioma research, correlation with tumor grade can be statistically assessed using methods described in published studies

• qPCR Validation:

  • Complement protein expression data with mRNA quantification

  • TaqMan assays have successfully been used to measure ATP1A1 mRNA levels

  • Report relative to appropriate housekeeping genes (studies have reported values below 5% compared to controls after successful knockdown)

• Knockdown Verification:

  • siRNA experiments targeting ATP1A1 have achieved reduction to below 5% mRNA level

  • Corresponding protein reduction reached approximately 35-39%

  • Temporal analysis shows stable knockdown for at least 72 hours

How can conflicting ATP1A1 antibody results be reconciled across different studies?

When faced with seemingly conflicting ATP1A1 antibody results:

• Epitope Mapping Analysis: Different antibodies targeting different regions of ATP1A1 may give varied results based on:

  • Protein conformation in different tissues

  • Post-translational modifications

  • Protein-protein interactions that may mask epitopes

• Sample Preparation Variations: Critical methodological differences that affect results include:

  • Heat treatment (37°C vs. boiling)

  • Detergent selection for membrane protein extraction

  • Fixation methods (critical for IHC/IF studies)

• Cell-Type Specific Expression: ATP1A1 expression varies significantly across:

  • Normal vs. cancer tissues

  • Stem cells vs. differentiated cells (shown in glioma studies)

  • Different regions within the same organ (e.g., kidney, brain)

• Quantification Method Standardization: For comparative studies:

  • Use the same detection system across all samples

  • Include positive control samples validated in previous studies

  • Apply statistical methods appropriate for the data distribution

• Experimental Validation: To resolve conflicts:

  • Use multiple antibodies targeting different epitopes

  • Apply complementary techniques (WB, IHC, IF)

  • Include genetic approaches (siRNA, CRISPR) to validate specificity