ATHB-54 Antibody

What is ATG4B antibody and what cellular processes does it target?

ATG4B antibody targets Autophagy Related 4B (ATG4B), also known as autophagin-1, APG4B, or AUTL1. This protein belongs to the peptidase C54 family and functions as a cysteine protease involved in autophagy. ATG4B is a cytoplasmic enzyme homologous to yeast Apg4 and is highly expressed in skeletal muscle, with lower expression in heart, liver, and pancreas. It's also widely expressed in tumor cell lines and has 5 isoforms produced by alternative splicing .

What applications is ATG4B antibody validated for?

The Anti-ATG4B Rabbit Polyclonal Antibody has been validated for several key applications:

• Western Blot (WB): Validated at dilutions of 1:200-1:2000 using human brain tissue

• Immunohistochemistry (IHC): Validated at dilutions of 1:20-1:200 using human pancreatic cancer tissue

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated for this application

• Other potential applications based on antibody characteristics include immunoprecipitation and immunofluorescence

What are the optimal storage conditions for research antibodies?

For ATG4B antibody specifically, the recommended storage is at -20°C. The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. These storage conditions are designed to maintain antibody stability and activity over time .

For research antibodies generally, it's important to:

• Avoid repeated freeze-thaw cycles

• Store aliquoted samples to prevent contamination

• Follow manufacturer's recommendations for long-term storage

• Monitor expiration dates and stability indicators

How can I optimize Western blot protocols for maximum sensitivity with research antibodies?

When performing Western blots with research antibodies like ATG4B antibody, consider these methodological optimizations:

• Sample preparation: Proper lysis buffers and protease inhibitors are essential; for gametocyte extracts as seen in Plasmodium research, heating samples at 56°C for 15 minutes in appropriate sample buffer is effective

• Protein separation: Use gradient gels (such as 4-12% Bis-Tris) for proteins with wide molecular weight ranges; non-reducing conditions may be necessary for certain antibodies to maintain epitope recognition

• Transfer optimization: Use systems like Trans-Blot Turbo with 0.22 μm nitrocellulose membranes for efficient protein transfer

• Blocking and antibody incubation: 5% skimmed milk in PBS works effectively for blocking; antibody concentration of 5 μg/ml is often suitable for primary incubation

• Detection: Use high-sensitivity ECL substrates like Clarity Western ECL for optimal visualization

What are the best approaches for immunoprecipitation with research antibodies?

For effective immunoprecipitation with research antibodies:

• Antibody coupling: Covalently link the antibody to tosyl-activated beads to create a stable antibody-bead complex that can withstand washing steps

• Sample preparation: Prepare clear lysates with appropriate lysis buffers that maintain protein structure while disrupting cellular components

• Antigen capture: Incubate the antibody-coupled beads with the sample lysate under gentle agitation to allow efficient antigen binding

• Elution optimization: Use appropriate elution conditions that release the antigen without contaminating the sample with antibody

• Analysis confirmation: Run eluted fractions on SDS-PAGE with silver staining to confirm specific antigen capture; include negative control antibodies (like anti-HIV gp120 mAb) to distinguish specific from non-specific binding

• Mass spectrometry follow-up: For unknown antigens, cut specific bands for mass spectrometry analysis against appropriate proteomic databases

How do framework mutations affect antibody performance, and how can this knowledge improve antibody development?

Framework mutations in antibodies can significantly impact their performance:

• Stability effects: Certain framework mutations like S54A can improve antibody stability by creating favorable hydrophobic interactions. Molecular dynamics simulations show that such mutations can decrease root mean square deviation (RMSD) and fluctuation (RMSF) values, indicating increased structural stability

• Immunogenicity impact: Framework mutations found in human antibody repertoires tend to reduce potential immunogenicity by removing T cell epitopes. Analysis shows increasing position-specific scoring matrix (PSSM) scores correlate with decreasing peptide:MHC-II binding affinity, which potentially decreases immunogenicity

• Antigen binding: Some framework mutations can reduce flexibility in complementarity-determining regions (CDRs), particularly CDRH2, which may enhance antigen binding by pre-organizing the binding site

• Development implications: Using human antibody repertoire-based position-specific scoring matrices (PSSMs) can help predict beneficial framework mutations early in antibody development, potentially reducing immunogenicity and improving stability

What computational methods can assess antibody stability and aid in antibody engineering?

Several computational approaches can evaluate and improve antibody stability:

What criteria should be used to validate antibody specificity for research applications?

When validating research antibodies for experimental use, consider these essential criteria:

• Multi-application validation: Confirm performance across multiple applications (WB, IHC, ELISA) as appropriate for the intended use

• Cross-reactivity testing: Verify species reactivity claims by testing with samples from all target species; for example, ATG4B antibody has reported reactivity with human, mouse, and rat samples

• Positive and negative controls: Use samples known to express high levels of the target (like skeletal muscle for ATG4B) and samples with no expected expression (like certain fetal tissues for ATG4B)

• Knockout/knockdown validation: Where possible, compare antibody reactivity between wild-type samples and those where the target protein has been eliminated or reduced

• Epitope characterization: Understand what region of the target protein the antibody recognizes, which helps interpret results when protein isoforms are present (ATG4B has 5 reported isoforms)

How can researchers distinguish between specific and non-specific binding in antibody-based techniques?

To differentiate specific from non-specific binding:

• Control antibodies: Include isotype-matched control antibodies that target irrelevant proteins; for example, using anti-HIV gp120 mAb as a control in immunoprecipitation experiments with gametocyte lysates

• Competing peptides: Pre-incubate antibodies with excess amounts of the target peptide/protein to block specific binding sites

• Band pattern analysis: In Western blots, compare observed band patterns with expected molecular weights and expression patterns; ATG4B, for instance, should show higher expression in skeletal muscle extracts compared to other tissues

• Multiple antibody validation: Use different antibodies targeting different epitopes of the same protein to confirm findings

• Mass spectrometry confirmation: For immunoprecipitation experiments, perform mass spectrometry on isolated protein bands to confirm their identity, as demonstrated in Plasmodium research

What are common causes of background signal in Western blots and how can they be addressed?

High background in Western blots can be caused by several factors:

• Insufficient blocking: Optimize blocking conditions; 5% skimmed milk in PBS is effective for many applications including ATG4B antibody experiments

• Antibody concentration: Titrate antibody dilutions; for ATG4B antibody, recommended dilutions range from 1:200 to 1:2000 for Western blots

• Secondary antibody issues: Ensure secondary antibodies are compatible and properly diluted; for human IgG detection, 1:5000 dilution of anti-human IgG-HRP has been effective

• Wash steps: Increase number or duration of washes between antibody incubations

• Buffer compatibility: Ensure the storage buffer of the antibody (PBS with 0.02% sodium azide and 50% glycerol for ATG4B antibody) doesn't interfere with the Western blot protocol

• Membrane choice: 0.22 μm nitrocellulose membranes provide good results for many proteins, including those in Plasmodium research

What strategies can improve signal detection for low-abundance targets?

For detecting low-abundance proteins with research antibodies:

• Sample enrichment: Consider immunoprecipitation before Western blotting to concentrate the target protein

• Enhanced detection substrates: Use high-sensitivity ECL substrates like Clarity Western ECL for improved signal

• Optimized antibody concentration: Higher antibody concentrations (up to 5 μg/ml) may be necessary for detecting low-abundance targets

• Improved transfer conditions: Systems like Trans-Blot Turbo can enhance protein transfer efficiency to the membrane

• Enhanced imaging: Use sensitive imaging systems like ImageQuant LAS4000 with longer exposure times as needed

• Reducing agents consideration: For some antibodies, non-reducing conditions may preserve epitopes better and improve detection sensitivity