ATP1B1 Human, Sf9

What is ATP1B1 Human recombinant protein expressed in Sf9 cells?

ATP1B1 Human recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 250 amino acids (63-303 a.a.) with a molecular mass of 29kDa. On SDS-PAGE, it typically appears at approximately 28-40 kDa due to glycosylation variations. The protein is expressed with a 6 amino acid His tag at the C-Terminus and purified using proprietary chromatographic techniques to achieve greater than 90% purity .

The protein represents the beta subunit of Na+/K+ ATPase, an essential membrane enzyme that maintains electrochemical gradients across cell membranes. The Sf9 insect cell expression system provides advantages for producing complex proteins requiring post-translational modifications while maintaining reasonable yields for research applications.

What are the optimal storage and handling conditions for ATP1B1 Human recombinant?

For maximum stability and activity retention, ATP1B1 Human recombinant protein requires specific storage conditions:

• Long-term storage: -20°C (recommended for maintaining protein integrity)

• Short-term usage: After reconstitution, store at 4°C for use within a few days

• Transport: Ship with wet ice to prevent degradation

• Critical consideration: Avoid freeze-thaw cycles which significantly reduce protein stability and activity

For reconstitution, use sterile buffers appropriate for your experimental system. Document the reconstitution date and maintain aliquots to prevent repeated freeze-thaw cycles if multiple experiments are planned over time.

How can I verify the purity and identity of ATP1B1 Human recombinant protein?

Implementing multiple verification methods ensures reliable experimental results:

• SDS-PAGE analysis:

  • Verify the expected molecular weight range (28-40 kDa)

  • Confirm purity exceeds 90% as indicated in product specifications

  • Look for appropriate band pattern reflecting glycosylation heterogeneity

• Western blot confirmation:

  • Use anti-His antibodies to detect the C-terminal tag

  • Employ specific anti-ATP1B1 antibodies for protein identity confirmation

  • Compare band patterns to positive controls when available

• Mass spectrometry:

  • Peptide mass fingerprinting for sequence verification

  • Analysis of post-translational modifications, particularly glycosylation patterns

  • Confirmation of molecular integrity and potential degradation products

What are appropriate experimental applications for ATP1B1 Human recombinant protein?

While the search results specify cell culture applications , ATP1B1 Human recombinant can be utilized in various research contexts:

• Structural and functional studies:

  • Investigation of Na+/K+ ATPase beta subunit structure

  • Analysis of subunit interactions within the Na+/K+ ATPase complex

  • Structure-function relationship studies through site-directed mutagenesis

• Interaction studies:

  • Protein-protein interaction analysis with alpha subunits

  • Identification of novel binding partners through pull-down assays

  • Characterization of interactions with potential inhibitors or modulators

• Cell-based assays:

  • Transfection into ATP1B1-deficient cell lines to restore function

  • Immunofluorescence studies to examine subcellular localization

  • Assessment of ion transport activity in reconstituted systems

• Antibody production and validation:

  • Generation of specific antibodies against human ATP1B1

  • Epitope mapping and immunological studies

  • Development of detection reagents for diagnostic applications

How should I design control experiments when working with ATP1B1 Human recombinant?

Robust experimental design requires appropriate controls to ensure result validity:

• Negative controls:

  • Buffer-only samples containing all components except ATP1B1

  • Heat-denatured ATP1B1 (95°C for 10 minutes) to confirm specificity

  • Non-specific proteins of similar size with His-tags for tag-related effects

• Positive controls:

  • Commercial Na+/K+ ATPase preparations when available

  • Native membrane preparations containing endogenous Na+/K+ ATPase

  • Previously validated batches of recombinant ATP1B1

• Specificity controls:

  • Known Na+/K+ ATPase inhibitors (e.g., ouabain) to confirm functional specificity

  • Alpha subunit co-expression to assess complex formation

  • Antibody blocking experiments to validate interaction specificity

What methodological considerations are important for ATP1B1 activity assays?

When assessing ATP1B1 functional activity, several methodological factors require attention:

• Buffer optimization:

  • pH optimization (typically pH 7.2-7.6)

  • Ionic composition (Na+, K+, Mg2+ concentrations)

  • ATP concentration and purity

  • Presence of appropriate detergents for membrane protein stability

• Detection systems:

  • Colorimetric assays for phosphate release

  • Coupled enzyme systems for ATP hydrolysis measurement

  • Fluorescence-based ion sensing for transport activity

  • Radiolabeled substrate approaches for highest sensitivity

• Data analysis:

  • Standard curve generation with known enzyme concentrations

  • Kinetic parameter determination (Km, Vmax)

  • Statistical analysis across multiple experimental replicates

  • Comparison with established literature values for validation

How does the expression system affect ATP1B1 protein characteristics?

The Sf9 baculovirus expression system imparts specific characteristics to recombinant ATP1B1:

• Glycosylation differences:

  • Sf9 cells produce high-mannose type N-glycans rather than complex mammalian patterns

  • This results in the observed molecular weight heterogeneity (28-40 kDa) on SDS-PAGE

  • Functional implications may include altered stability and binding kinetics

• Protein folding and conformation:

  • Insect cells generally provide appropriate chaperone systems for human protein folding

  • The C-terminal His-tag may influence terminal domain folding or interactions

  • Potential differences in disulfide bond formation compared to mammalian systems

• Comparative considerations:

  • For studies requiring native-like glycosylation, mammalian expression systems may be preferable

  • For structural studies, bacterial expression with refolding protocols might yield higher quantities

  • For functional studies, comparing results across expression systems provides valuable insights

How can I address experimental variability when working with ATP1B1?

Managing experimental variability requires systematic approaches:

• Protein stability monitoring:

  • Implement regular quality control testing of stored aliquots

  • Track protein activity over time under different storage conditions

  • Establish acceptance criteria for experimental use

• Assay standardization:

  • Develop standard operating procedures (SOPs) for all experimental protocols

  • Include internal controls in every experimental run

  • Calculate and monitor inter-assay coefficients of variation

• Data interpretation strategies:

  • Employ statistical methods appropriate for your experimental design

  • Consider multivariate analysis when multiple factors affect outcomes

  • Implement blinding procedures where appropriate to reduce bias

What approaches can resolve conflicting experimental results with ATP1B1?

When faced with contradictory findings:

• Systematic troubleshooting approach:

  • Verify protein integrity through orthogonal methods

  • Examine buffer components for potential interfering substances

  • Consider batch-to-batch variation in recombinant protein

• Experimental parameter analysis:

  • Create a comprehensive table documenting all variables across experiments

  • Systematically alter single variables to identify critical factors

  • Consider temperature, pH, ionic strength, and protein concentration effects

• Resolution strategies:

  • Implement multiple detection methodologies for the same endpoint

  • Conduct dose-response experiments to identify potential threshold effects

  • Consult literature for similar contradictions and resolution approaches

How can ATP1B1 Human recombinant be utilized in structure-function studies?

Structure-function analysis with ATP1B1 can employ several complementary approaches:

• Mutagenesis strategies:

  • Site-directed mutagenesis of key residues based on structural predictions

  • Creation of chimeric constructs with other beta subunit isoforms

  • Terminal or internal deletion constructs to map functional domains

• Structural analysis methods:

  • Circular dichroism (CD) spectroscopy for secondary structure assessment

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • Small-angle X-ray scattering (SAXS) for solution structure information

• Functional correlation:

  • Systematic correlation of structural modifications with activity measurements

  • Assessment of alpha/beta subunit assembly efficiency with structural variants

  • Evaluation of glycosylation site mutations on protein stability and function

What considerations are important when using ATP1B1 in drug discovery research?

ATP1B1 as a component of Na+/K+ ATPase presents opportunities for therapeutic research:

• Screening methodology:

  • Binding assays with labeled compounds or fragments

  • Activity-based screening using ATP hydrolysis as readout

  • Thermal shift assays to identify stabilizing compounds

• Pharmacogenetic implications:

  • Consideration of known ATP1B1 genetic variants in drug response

  • Potential incorporation into personalized medicine approaches

  • Connection to broader pharmacogenetic frameworks as seen with other therapeutic targets

• Data interpretation challenges:

  • Distinguishing direct effects on beta subunit from alpha subunit mediated effects

  • Accounting for species differences in drug response

  • Translating in vitro findings to cellular and in vivo contexts

How can ATP1B1 Human recombinant protein research contribute to understanding disease mechanisms?

ATP1B1 research offers insights into various pathological conditions:

• Relevant disease associations:

  • Cardiovascular disorders involving ion transport dysregulation

  • Neurological conditions affected by ion gradients

  • Potential roles in cancer cell metabolism and survival

• Experimental approaches:

  • Comparison of wild-type and disease-associated variants

  • Investigation of post-translational modification patterns in disease states

  • Analysis of protein-protein interactions under physiological and pathological conditions

• Translational potential:

  • Development of biomarkers based on ATP1B1 status

  • Target validation studies for therapeutic development

  • Insights into ion transport mechanisms relevant to drug action and toxicity

How can I optimize protein yield and quality when working with ATP1B1 in Sf9 cells?

Maximizing recombinant ATP1B1 production requires optimization at multiple levels:

• Expression parameters:

  • Viral titer optimization through systematic testing

  • Cell density at infection (typically 1-2 × 10^6 cells/ml)

  • Harvest timing determination through time-course experiments

  • Temperature and media composition adjustments

• Extraction optimization:

  • Detergent selection and concentration for membrane protein solubilization

  • Lysis buffer composition including protease inhibitors

  • Incubation time and temperature during solubilization

  • Centrifugation parameters for optimal clarification

• Purification strategy:

  • Affinity chromatography conditions leveraging the C-terminal His tag

  • Washing stringency to balance purity and yield

  • Elution conditions to maintain protein stability

  • Secondary purification steps if higher purity is required

What analytical methods are most informative for ATP1B1 characterization?

Comprehensive characterization requires multiple analytical approaches:

• Purity assessment:

  • SDS-PAGE with various staining methods (Coomassie, silver, fluorescent)

  • Size exclusion chromatography to detect aggregates and oligomeric states

  • Capillary electrophoresis for high-resolution analysis

• Functional characterization:

  • ATPase activity assays with varying substrate concentrations

  • Binding assays with alpha subunits and other interacting partners

  • Thermal stability assessment under various buffer conditions

• Structural evaluation:

  • Mass spectrometry for accurate molecular weight determination

  • Glycan analysis using specific glycosidases and lectin binding

  • Limited proteolysis to identify stable domains and flexible regions

How should I address solubility and stability challenges with ATP1B1?

As a membrane-associated protein, ATP1B1 presents specific handling challenges:

• Solubility enhancement:

  • Systematic screening of detergent types and concentrations

  • Addition of lipids or lipid-like molecules to stabilize native conformation

  • Buffer optimization including pH, ionic strength, and stabilizing additives

  • Consideration of protein concentration effects on aggregation propensity

• Storage stability:

  • Strict adherence to recommended storage conditions (-20°C long-term, 4°C short-term)

  • Addition of glycerol (typically 10-20%) or other cryoprotectants

  • Aliquoting to appropriate volumes to eliminate freeze-thaw cycles

  • Monitoring stability through activity assays over time

• Experimental stability:

  • Temperature control during experimental procedures

  • Minimizing exposure to air/surface interfaces

  • Addition of reducing agents if disulfide scrambling is a concern

  • Consideration of metal chelators if metal-catalyzed oxidation occurs