ATP5J2 Antibody

What is ATP5J2 and why is it significant for mitochondrial research?

ATP5J2 (also known as ATP5MF) is part of the complex F0 domain of mitochondrial ATP synthase. This enzyme produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain. The ATP synthase complex consists of two primary components: the soluble catalytic core (F1) and the membrane-spanning component (F0), which comprises the proton channel. ATP5J2 specifically encodes the f subunit of the F0 complex, and there are two isoforms produced by alternative splicing .

The significance of ATP5J2 lies in its critical role in cellular energy production and its potential involvement in age-associated diseases, including dementia. Research has identified ATP synthase as a shared drug target for aging and age-associated dementia, making ATP5J2 antibodies essential tools for investigating mitochondrial function in both normal and disease states .

What applications are ATP5J2 antibodies validated for?

According to the search results, ATP5J2 antibodies have been validated for multiple research applications:

It's important to note that optimal dilutions/concentrations should be determined by the end user based on specific experimental conditions .

How should ATP5J2 antibodies be stored to maintain optimal activity?

Storage recommendations for ATP5J2 antibodies vary slightly depending on the manufacturer, but generally follow these guidelines:

• Short-term storage (up to one month): Store at 4°C

• Long-term storage: Store at -20°C or -80°C

• Avoid repeated freeze/thaw cycles to maintain antibody integrity

• Some formulations contain glycerol (e.g., 50% glycerol with PBS)

• Many formulations contain 0.02% sodium azide as a preservative

For the specific Invitrogen ATP5J2 Polyclonal Antibody, the storage recommendation is: "Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles" .

How should I optimize Western blot conditions when using ATP5J2 antibodies?

When optimizing Western blot protocols for ATP5J2 detection, consider the following:

• Molecular weight considerations: ATP5J2 is a relatively small protein with a calculated molecular weight of approximately 10.9 kDa , with observed molecular weight around 11 kDa . Use appropriate gel percentage (12-15% acrylamide) for better resolution of small proteins.

• Sample preparation:

  • Ensure complete denaturation of mitochondrial proteins

  • Consider enriching mitochondrial fractions for better detection

  • Use appropriate lysis buffers containing protease inhibitors

• Transfer conditions:

  • Optimize transfer time and voltage for small proteins

  • Consider using PVDF membranes with smaller pore sizes

  • Use wet transfer rather than semi-dry for small proteins

• Antibody concentration:

  • Start with manufacturer's recommended dilutions (1:5000-1:50000)

  • Perform antibody titration experiments to determine optimal concentration

  • Extended primary antibody incubation at 4°C overnight may improve results

• Positive controls:

  • Include mitochondrial-enriched samples from tissues known to express ATP5J2 (heart tissue shows good expression)

The ATP5J2 antibody from Proteintech (15842-1-AP) has been shown to detect ATP5J2 in mouse and rat heart tissue samples by Western blot .

What are the best practices for immunohistochemistry (IHC) using ATP5J2 antibodies?

For optimal IHC results with ATP5J2 antibodies, consider these methodological recommendations:

• Tissue preparation:

  • Proper fixation is critical (typically 10% neutral buffered formalin)

  • Consider antigen retrieval methods to expose epitopes that may be masked during fixation

  • For mitochondrial proteins, citrate buffer (pH 6.0) heat-induced epitope retrieval is often effective

• Antibody dilution:

  • Start with recommended dilutions (1:50-1:200 or 1:500-1:1000 )

  • Optimize by testing several dilutions on positive control tissues

• Controls:

  • Include tissues known to express ATP5J2 (such as lung tissue)

  • Include a negative control by omitting primary antibody

  • Consider using siRNA knockdown tissues/cells as specificity controls

• Detection systems:

  • For low abundance targets like ATP5J2, amplification systems may improve sensitivity

  • Consider tyramide signal amplification or polymer-based detection systems

• Counterstains:

  • Use mitochondrial counterstains to confirm localization

  • Consider dual staining with other mitochondrial markers

Validation data shows successful IHC detection of ATP5J2 in paraffin-embedded human lung carcinoma tissue at 1:100 dilution .

How can ATP5J2 antibodies be used to study mitochondrial ATP synthase inhibition and neuroprotection?

Research has revealed that ATP synthase inhibition may confer neuroprotective effects in certain contexts. ATP5J2 antibodies can be valuable tools for investigating this phenomenon:

• Experimental approaches:

  • Combine ATP5J2 antibodies with inhibitors (such as J147) to study mechanisms of ATP synthase modulation

  • Use ATP5J2 antibodies to measure protein levels after siRNA-mediated knockdown of ATP5A

  • Compare ATP5J2 expression in models of neurotoxicity with and without neuroprotective interventions

• Relevant methodologies:

  • Co-immunoprecipitation to study protein-protein interactions in the ATP synthase complex

  • Immunofluorescence to track subcellular localization changes during stress

  • Western blotting to quantify expression changes

Research shows that ATP synthase inhibition protects from neurotoxic insults. When ATP5A (a catalytic subunit of ATP synthase) is knocked down, it provides protection against amyloid proteotoxicity, glutamate-induced glutathione depletion (oxytosis), and iodoacetic acid (IAA)-induced energy depletion . ATP5J2 antibodies can be used to study whether these protective effects involve changes in ATP5J2 expression or localization.

How can I investigate the interaction between Connexin 43 and ATP5J2 using available antibodies?

Research has identified an interaction between Connexin 43 (Cx43) and ATP5J2 that regulates mitochondrial ATP generation. To investigate this interaction:

• Co-immunoprecipitation (Co-IP):

  • Use ATP5J2 antibodies to immunoprecipitate the protein complex and probe for Cx43

  • Alternatively, use Cx43 antibodies for IP and probe for ATP5J2

  • A validated approach demonstrated Cx43 detection when anti-GFP antibody against ATP5J2-GFP was used as immunoprecipitating antibody

• Förster Resonance Energy Transfer (FRET):

  • This technique has successfully confirmed co-localization and interaction between mtCx43 and ATP5J2

  • Requires fluorescent labeling of both proteins

• Protein pull-down assays:

  • Use GST-tagged Cx43 CT (C-terminal) immobilized on glutathione-agarose beads to pull down ATP5J2-GFP

  • This approach demonstrated direct interaction between Cx43 CT and ATP5J2 in the intermembrane space

• Molecular dynamics simulations:

  • In silico modeling of the interaction interface between Cx43 and ATP5J2

  • Identification of critical amino acid residues involved in binding

• Functional assays:

  • Use ATP determination assays to measure the effect of Cx43 knockdown on ATP production

  • Compare ATP levels in whole-cell, mitochondrial, and cytosolic fractions

The research demonstrated that Cx43 knockdown decreased ATP production dramatically, and ATP determination confirmed reduced ATP in whole-cell lysates, isolated mitochondria, and cytosolic components of Cx43KD cells compared to controls .

What are the considerations for studying ATP5J2 in subcellular fractionation experiments?

When conducting subcellular fractionation to study ATP5J2 localization:

• Mitochondrial isolation protocols:

  • Use gentle homogenization to preserve mitochondrial integrity

  • Differential centrifugation or density gradient methods may be employed

  • Commercial mitochondrial isolation kits are available but may need optimization

• Purity assessment:

  • Use markers for mitochondrial fractions (e.g., COXIV, VDAC)

  • Check for contamination from other organelles

  • Use multiple mitochondrial markers to verify results

• Subfractionation considerations:

  • ATP5J2 is located in the F0 domain (membrane-embedded portion)

  • Consider separating mitochondrial membrane and matrix fractions

  • Gentle detergent treatment can separate inner and outer mitochondrial membranes

• Antibody selection:

  • Choose antibodies that recognize epitopes preserved during fractionation

  • Consider antibodies that can distinguish between isoforms if relevant

• Data analysis:

  • Quantify ATP5J2 enrichment in fractions relative to total protein

  • Compare with known mitochondrial markers

  • Consider both protein abundance and enzyme activity measurements

Recent research has also discovered that ATP synthase complexes may be found not only in mitochondria but also delivered to the cell surface along microtubules . This finding adds complexity to localization studies and suggests researchers should consider both mitochondrial and potential extramitochondrial pools of ATP5J2.

How can I address weak or nonspecific signals when using ATP5J2 antibodies in Western blots?

When troubleshooting Western blot issues with ATP5J2 antibodies:

• For weak signals:

  • Increase primary antibody concentration

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection systems (e.g., enhanced chemiluminescence)

  • Enrich for mitochondrial proteins in your sample

  • Increase protein loading while ensuring even transfer

  • Consider using PVDF membranes instead of nitrocellulose for small proteins

• For nonspecific bands:

  • Increase blocking time or concentration of blocking agent

  • Add 0.1-0.5% Tween-20 in the washing buffer

  • Decrease primary antibody concentration

  • Pre-adsorb the antibody with non-specific proteins

  • Use a more specific secondary antibody

  • Increase the stringency of washes

• For unexpected molecular weight bands:

  • Consider post-translational modifications

  • Check for potential isoforms (ATP5J2 has 2 isoforms from alternative splicing)

  • Consider protein degradation (add fresh protease inhibitors)

  • Check for potential protein complexes if using non-reducing conditions

• For inconsistent results between experiments:

  • Standardize protein extraction protocols

  • Use fresh antibody aliquots to avoid freeze/thaw cycles

  • Consider using recombinant ATP5J2 as a positive control

  • Standardize exposure times between experiments

The expected molecular weight of ATP5J2 is approximately 10.9 kDa , but the observed molecular weight may be around 11 kDa .

What controls should be included when validating a new batch of ATP5J2 antibody?

To properly validate a new batch of ATP5J2 antibody:

• Positive controls:

  • Tissues/cells known to express ATP5J2 (heart tissue shows good expression)

  • Recombinant ATP5J2 protein

  • Previously validated sample with confirmed ATP5J2 expression

• Negative controls:

  • ATP5J2 knockdown cells (siRNA or CRISPR)

  • Tissues with minimal ATP5J2 expression

  • Secondary antibody only (no primary antibody)

  • Pre-immune serum (for polyclonal antibodies)

• Specificity controls:

  • Competition assay with immunizing peptide

  • Comparison with other validated ATP5J2 antibodies

  • Testing across multiple species if the antibody claims cross-reactivity

• Technical validation:

  • Antibody titration to determine optimal concentration

  • Testing different blocking agents and conditions

  • Validation across multiple techniques (WB, IHC, IF)

• Lot-to-lot comparison:

  • Run side-by-side comparison with previously validated lot

  • Document and compare signal intensity, background, and specificity

The ATP5J2 antibody from Fisher Scientific is reported to be affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen with purity > 95% (by SDS-PAGE) , which provides a baseline quality standard.

How can ATP5J2 antibodies be utilized in aging and neurodegenerative disease research?

ATP5J2 antibodies can be valuable tools for investigating the role of mitochondrial dysfunction in aging and neurodegenerative diseases:

• Comparative expression analysis:

  • Compare ATP5J2 expression levels in young versus aged tissues

  • Analyze expression changes in neurodegenerative disease models

  • Examine post-translational modifications in disease states

• Drug target validation:

  • Research has identified ATP synthase as a shared drug target for aging and age-associated dementia

  • Use ATP5J2 antibodies to study the effects of compounds like J147 that target ATP synthase

  • Investigate drug-induced changes in ATP synthase complex assembly and activity

• Mechanistic studies:

  • Investigate how ATP5J2 contributes to mitochondrial dysfunction in disease

  • Study the relationship between ATP synthase inhibition and neuroprotection

  • Examine ATP5J2's role in modulating mitochondrial membrane potential (Δψm)

• Therapeutic development:

  • Screen for compounds that modulate ATP5J2 function

  • Use antibodies to validate target engagement

  • Monitor treatment-induced changes in ATP5J2 expression or PTMs

Research has shown that partial inhibition of ATP synthase activity by J147 with an EC50 of 20 nM (23.6 ± 3.4% inhibition at saturation) has neuroprotective effects . ATP5J2 antibodies can help elucidate whether this subunit plays a specific role in the neuroprotective mechanism.

What are the considerations for studying ATP5J2 interactions with other proteins in the ATP synthase complex?

When investigating protein-protein interactions involving ATP5J2:

• Co-immunoprecipitation approaches:

  • Use anti-ATP5J2 antibodies to pull down the entire complex

  • Probe for other ATP synthase subunits or interacting proteins

  • Consider cross-linking prior to IP to capture transient interactions

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to ATP5J2

  • Mass spectrometry to identify labeled proteins

• Structural biology considerations:

  • Cryo-EM studies of the ATP synthase complex

  • Use antibodies to validate structural models

  • Consider epitope accessibility within the assembled complex

• Interaction dynamics:

  • Study how interactions change under different physiological conditions

  • Examine how drug treatments affect complex assembly

  • Investigate isoform-specific interactions

• Functional consequences:

  • Correlate interaction changes with ATP synthase activity

  • Study how mutations affect protein-protein interactions

  • Examine the impact of post-translational modifications

Recent research has demonstrated interactions between ATP5J2 and Connexin 43 (Cx43), suggesting that ATP5J2 interactions extend beyond the ATP synthase complex itself . This highlights the importance of considering both canonical and non-canonical interaction partners.

How can photolabeling approaches be combined with ATP5J2 antibodies for dynamic protein tracking?

Photolabeling combined with ATP5J2 antibodies offers powerful approaches for tracking protein dynamics:

• Photoactivatable GFP fusion proteins:

  • ATP5B-paGFP has been successfully used to track ATP synthase dynamics

  • Similar ATP5J2-paGFP constructs could be developed

  • Validate fusion protein incorporation into ATP synthase complex using anti-ATP5J2 antibodies

• Pulse-chase experiments:

  • Activate paGFP in specific cellular regions

  • Track movement of labeled proteins over time

  • Use antibodies to confirm identity and interactions

• Dual-color imaging:

  • Combine ATP5J2-paGFP with MitoTracker or other organelle markers

  • Track movement between mitochondria and other cellular locations

  • Study the surprising finding that ATP synthase complexes can be delivered to the cell surface

• Super-resolution microscopy:

  • Use photoactivatable fluorophores for single-molecule localization microscopy

  • Combine with immunofluorescence using ATP5J2 antibodies

  • Achieve nanoscale resolution of ATP synthase complex distribution

• Quantitative analysis:

  • Measure protein turnover rates

  • Calculate diffusion coefficients

  • Determine interaction kinetics

Research using ATP5B-paGFP has shown that the ATP synthase complex is first assembled in mitochondria and subsequently delivered to the cell surface along microtubules . Similar approaches could be applied to study ATP5J2 dynamics specifically.