GBE1 Antibody, Biotin conjugated

What is GBE1 and what is its biological significance in glycogen metabolism?

GBE1 (glucan (1,4-alpha-), branching enzyme 1) is an essential enzyme that catalyzes the transfer of alpha-1,4-linked glucosyl units from the outer end of a glycogen chain to an alpha-1,6 position on the same or neighboring glycogen chain . This branching activity is crucial for creating the globular and branched structure of glycogen, which significantly increases its solubility by forming a hydrophilic surface . The physiological importance of this branching extends to reducing intracellular osmotic pressure, thereby maintaining cellular homeostasis . GBE1 functions in coordination with glycogenin and glycogen synthase to facilitate the entire glycogen biosynthetic process . Its role is particularly critical in tissues with high energy demands, such as liver and muscle, where glycogen serves as a primary energy storage molecule .

What is the molecular weight of GBE1 protein and how does this affect antibody detection?

GBE1 has a calculated molecular weight of approximately 80 kDa (80.474 kDa specifically) , though the observed molecular weight may vary slightly depending on the detection method and sample preparation. The search results indicate observed molecular weights of 72-80 kDa . This variation is important for researchers to consider when validating antibody specificity. The full-length protein consists of 702 amino acids , but it's worth noting that the mature form may be processed to approximately 75 kDa . When designing experiments using GBE1 antibodies, researchers should account for these potential variations to accurately interpret Western blot results and avoid false negatives or misidentification of bands.

What applications are biotin-conjugated GBE1 antibodies suitable for?

The biotin conjugation provides versatility through compatibility with various streptavidin-conjugated detection systems, making these antibodies particularly valuable for multiplexing experiments.

What dilutions are recommended for biotin-conjugated GBE1 antibodies in different applications?

While specific dilutions for the biotin-conjugated format aren't directly stated in the search results, researchers can extrapolate from recommendations for unconjugated GBE1 antibodies with appropriate modifications to account for the biotin conjugation:

It is critically important to note that these dilutions should serve as starting points, and researchers should conduct titration experiments to determine optimal conditions for their specific experimental setup. As stated in the search results, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

How should samples be prepared for optimal detection of GBE1 using biotin-conjugated antibodies?

Effective sample preparation is crucial for successful detection of GBE1. Based on the validation data from the search results, the following preparation methods have proven successful:

For Western Blot:

• Extract proteins using a compatible lysis buffer (RIPA or similar buffer systems)

• Load 20-30 μg of total protein per lane as demonstrated in published validation data

• Separate proteins on 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving) for 2-3 hours as per validated protocols

• Transfer to nitrocellulose membrane at approximately 150 mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• For biotin-conjugated antibodies, utilize streptavidin-HRP in the detection step

For ELISA:

• Coat plates with target protein or cell/tissue lysates

• After blocking, apply biotin-conjugated GBE1 antibody at optimized dilution

• Detect using streptavidin-conjugated enzyme system

• Develop signal using compatible substrate

The search results indicate successful detection in multiple sample types including human fetal liver lysate, PC-3 lysate, and various tissue samples from human, mouse, and rat origins , providing researchers with validated options for positive controls.

What are appropriate positive and negative controls for GBE1 antibody validation experiments?

Selecting appropriate controls is essential for validating antibody specificity and ensuring experimental rigor:

Positive Controls (validated in search results):

• Human samples: L02 cells, HeLa cells, PC-3 cells, RT4 cells, U251 cells, fetal liver lysate, skeletal muscle tissue

• Mouse samples: Liver tissue

• Rat samples: Liver tissue

Negative Controls:

• GBE1 knockout or knockdown samples as referenced in published applications

• Pre-adsorption with immunizing peptide/protein (the search results indicate the immunogen was recombinant human GBE1 protein, positions H79-R515 or 1-300aa)

• Isotype control antibody at equivalent concentration

• Secondary antibody-only control to assess non-specific binding

For definitive validation, researchers should consider employing CRISPR/Cas9-mediated GBE1 knockout or siRNA-mediated knockdown approaches, which have been documented in at least one publication referenced in the search results .

How can I distinguish between potential isoforms or post-translational modifications of GBE1?

Distinguishing between different forms of GBE1 requires careful experimental design and analysis:

• High-resolution gel systems: Use gradient gels (4-20%) to achieve better separation of closely migrating bands

• 2D gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate based on both charge and size

• Mass spectrometry: For definitive identification of specific isoforms or modifications

• Targeted antibodies: The search results indicate that the full GBE1 sequence can be processed into a mature form of 75 kDa . Consider using antibodies targeting different epitopes (N-terminal vs. C-terminal) to distinguish processing variants:

  • ABIN7142069 targets amino acids 1-300

  • Other antibodies target regions like AA 527-556, AA 605-702, or the middle region (AA 534-564)

When analyzing Western blot results, note that the observed molecular weight (72-80 kDa) may reflect tissue-specific processing or post-translational modifications. The calculated molecular weight is 80 kDa (702 amino acids), but observed weights may vary .

What factors might affect signal strength and specificity when using biotin-conjugated GBE1 antibodies?

Several factors can impact the performance of biotin-conjugated antibodies:

• Endogenous biotin: Tissues with high endogenous biotin (like liver, kidney, brain) may produce background signal. Consider using avidin/biotin blocking kits prior to antibody application.

• Biotin-to-antibody ratio: Optimal conjugation preserves antibody affinity while providing sufficient biotin for detection. Over-biotinylation can reduce antibody specificity.

• Storage conditions: The biotin conjugate should be stored as recommended (typically -20°C) to maintain stability. As noted in the search results: "Store at -20°C. Stable for one year after shipment."

• Buffer components: The search results indicate the storage buffer contains "PBS with 0.02% sodium azide and 50% glycerol pH 7.3" . Sodium azide can inhibit HRP activity, so thorough washing is essential before detection steps.

• Detection system quality: Use high-quality, fresh streptavidin conjugates for optimal signal-to-noise ratio.

To optimize signal-to-noise ratio, researchers should:

• Titrate primary antibody concentration

• Optimize incubation times and temperatures

• Use appropriate blocking reagents

• Include thorough washing steps

• Consider signal amplification systems for low-abundance targets

What are common pitfalls when using biotin-conjugated antibodies for GBE1 detection and how can they be addressed?

Several challenges may arise when working with biotin-conjugated GBE1 antibodies:

When troubleshooting, remember that the observed molecular weight of GBE1 may range from 72-80 kDa , and the mature form may be processed to approximately 75 kDa . This information is crucial when evaluating Western blot results.

How does species cross-reactivity affect experimental design when using biotin-conjugated GBE1 antibodies?

The search results provide valuable information about species reactivity that should guide experimental design:

• Confirmed reactivity: The biotin-conjugated antibody (ABIN7142069) is specifically validated for human samples . Other GBE1 antibodies have demonstrated reactivity with human, mouse, and rat samples .

• Sequence homology: Search result notes that "Human GBE1 shares 92.4% amino acid (aa) sequence identity with mouse GBE1," suggesting high conservation across mammalian species.

• Epitope consideration: The biotin-conjugated antibody targets amino acids 1-300 of GBE1 . Researchers working with non-human samples should align this region across species to predict potential cross-reactivity.

When working with species not explicitly validated:

• Perform preliminary validation experiments

• Include appropriate positive and negative controls

• Consider epitope sequencing alignment analysis

• Start with applications requiring less stringent specificity (like Western blot) before moving to more demanding applications (like IHC)

The search results show successful detection in multiple species, with validated reactivity in human cell lines (L02, HeLa, PC-3, RT4, U251) and tissues from human, mouse, and rat .

What are the optimal storage conditions for maintaining biotin-conjugated GBE1 antibody activity?

Proper storage is critical for maintaining antibody functionality. Based on the search results:

• Temperature: Store at -20°C for long-term storage . The search results indicate stability for one year after shipment under these conditions .

• Formulation: The antibody may be provided in different forms:

  • Liquid format containing "PBS with 0.02% sodium azide and 50% glycerol pH 7.3"

  • Lyophilized format that requires reconstitution

• Aliquoting: For 20μl sizes, aliquoting is noted as "unnecessary for -20°C storage" , but for larger volumes, aliquoting is generally recommended to avoid repeated freeze-thaw cycles.

• Reconstitution (if lyophilized): Add the specified volume of distilled water to achieve the desired concentration. For example, "Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml" .

• Working solution stability: Once diluted for use, store at 4°C for short-term use (up to one month) .

These storage guidelines are essential for maintaining antibody performance and ensuring reproducible results across experiments.

How can the stability of the biotin conjugation be monitored over time?

Monitoring the stability of biotin conjugation is important for ensuring consistent experimental results:

• Functional testing: Periodically test the antibody in a standardized ELISA or dot blot with known positive controls to assess any decline in signal intensity.

• Activity comparison: Maintain a reference aliquot stored optimally and compare new experiments to this standard.

• Avidin-binding assay: A simple test using immobilized avidin and detection of the antibody portion can assess whether the biotin component remains functional.

• Visual inspection: Biotin-conjugated antibody solutions should remain clear. Cloudiness or precipitation may indicate degradation.

• Documentation: Maintain records of antibody performance over time, including lot number, date received, and signal intensity in standard assays.

If diminished performance is observed, consider acquiring a new lot of antibody, as the biotin conjugation has likely degraded. Most manufacturers, including those referenced in the search results, provide guidance on expected shelf life under recommended storage conditions.