Customize pas-7 Antibody

Description

Antigen Characteristics

PAS-7 (Proteasome subunit alpha type-3) is the C. elegans ortholog of the human PSMA3/α3 proteasome subunit.

Molecular Weight: Predicted: 28 kDa; Observed: 28 kDa on Western blots .
Function: Essential for ubiquitin-dependent protein degradation, critical for cellular homeostasis and stress response .

Antibody Development

Developed by Nonet, M.L., Hadwiger, G., and Dour, S. at Washington University Medical School, the CePAS7 antibody was generated using:

Immunogen: Recombinant His6-tagged PAS-7 full-length fusion protein .
Host Species: Mouse (IgG1 isotype) .
Clonality: Monoclonal (clone CePAS7) .

Western Blot

Detects a single band at 28 kDa in C. elegans lysates, confirming specificity .
Validated in studies analyzing proteasome composition and function .

Immunofluorescence

Localizes PAS-7 to the cytoplasm and nuclei in C. elegans whole-mount preparations .
Used to study proteasome dynamics during developmental stages .

Key Publications

Study Focus	Findings	Citation
Proteasome subunit analysis	Identified PAS-7 as essential for larval development and stress response	Hadwiger et al., 2010
Protein degradation pathways	Linked PAS-7 to ubiquitin-mediated degradation in germline cells	Cheng et al., 2010

Comparative Notes

Species Specificity: Unlike cross-reactive PAX7 antibodies (e.g., DSHB PAX7 targeting satellite cells in vertebrates) , CePAS7 is exclusive to C. elegans .
Structural Distinction: PAS-7 is unrelated to PAX7, a transcription factor regulating myogenesis .

This antibody remains a critical tool for studying proteasome biology in C. elegans, with standardized protocols ensuring reproducibility across studies . For methodology sections, cite:

CePAS7 was deposited to the DSHB by Nonet, M.L. / Hadwiger, G. / Dour, S. (DSHB Hybridoma Product CePAS7) .

Product Specs

Buffer

Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

pas-7 antibody; ZK945.2 antibody; Proteasome subunit alpha type-3 antibody; EC 3.4.25.1 antibody; Proteasome subunit alpha 7 antibody

Target Names

pas-7

Uniprot No.

Q09583

Target Background

Function

The proteasome is a multi-catalytic proteinase complex. It is characterized by its ability to cleave peptides with arginine, phenylalanine, tyrosine, leucine, and glutamate residues adjacent to the leaving group at neutral or slightly basic pH. The proteasome exhibits ATP-dependent proteolytic activity.

Database Links

KEGG: cel:CELE_ZK945.2

STRING: 6239.ZK945.2.2

UniGene: Cel.14673

Protein Families

Peptidase T1A family

Subcellular Location

Cytoplasm. Nucleus.

Q&A

Basic Research Questions

How to validate PAS-7 antibody specificity in C. elegans models?

Perform parallel experiments using:

Western blot: Compare wild-type vs. pas-7 knockout lysates. Expect a 28 kDa band only in wild-type samples .
Immunofluorescence: Use tissues with known PAS-7 expression (e.g., neuronal cells) and include isotype controls .
Competition assays: Pre-incubate antibody with recombinant PAS-7 protein (1:5 molar ratio) to confirm signal loss .

What are optimal antibody concentrations for different applications?

Application	Starting Concentration	Buffer System
Western Blot	0.2-0.5 μg/ml	Tris-buffered saline + 0.1% Tween
Immunofluorescence	2-5 μg/ml	PBS + 1% BSA
Adjust concentrations based on signal-to-noise ratio, using 3-5 technical replicates per condition.

Advanced Research Challenges

How to resolve contradictory results in proteasome assembly studies using PAS-7 antibodies?

Case example: Discrepancies in 26S proteasome quantification may arise from:

Factor	Solution
Sample preparation	Use fresh lysates with 5 mM ATP in lysis buffer to maintain proteasome integrity
Epitope accessibility	Combine chemical crosslinking (1% formaldehyde, 10 min) with gentle sonication
Antibody batch variation	Include positive control lysates in every experiment

What strategies improve PAS-7 detection in protein degradation kinetics studies?

Pulse-chase design: Incorporate MG132 (10 μM) to block proteasomal activity during pulse phase
Multiplex detection: Combine PAS-7 antibody with fluorogenic proteasome substrates (e.g., Suc-LLVY-AMC) for real-time activity correlation
Data normalization: Use β5 subunit levels as internal reference for proteasome quantification

Methodological Considerations

How to design experiments investigating PAS-7/Wnt signaling crosstalk?

Establish baseline: Map PAS-7 expression gradients in Wnt-activated vs. inhibited conditions (use CHIR99021/XAV939 treatments)
Employ co-immunoprecipitation with:
- Anti-Frizzled-7 antibodies (1:100 dilution)
- SMAD4 co-detection (1:50 dilution, 24 hr incubation)
Validate findings using tissue-specific RNAi with ≥3 biological replicates

What controls are essential for developmental studies using PAS-7 antibodies?

Control Type	Implementation
Biological	pas-7(RNAi) and wild-type synchronized larvae
Technical	Spiked-in human 20S proteasome standards (10 ng/μL)
Procedural	Parallel samples without primary antibody

Data Interpretation Framework

How to analyze PAS-7 antibody performance across research groups?

Create standardized metrics:

Metric	Calculation	Acceptable Range
Signal Specificity	(Knockout signal/WT signal) × 100	≤15%
Lot Consistency	CV across 3 antibody batches	≤20%
Dynamic Range	Linear regression R² (1-100 ng load)	≥0.98

What emerging techniques complement PAS-7 antibody applications?

Cryo-EM sample prep: Use PAS-7 localization patterns to guide particle picking in 26S proteasome reconstructions
Single-cell proteomics: Combine antibody staining with mass cytometry (CyTOF) using metal-tagged secondary reagents
Live imaging: Develop PAS-7 intrabodies fused with pH-sensitive fluorophores (e.g., pHluorin)

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pas-7 Antibody