BOLA1 Antibody

What is BOLA1 protein and what are its main functions?

BOLA1 (bolA homolog 1) is a mitochondrial protein belonging to the BolA protein family, which is widespread among eukaryotes and bacteria. The calculated molecular weight of BOLA1 is approximately 14 kDa . As a mitochondrial protein, BOLA1 performs several critical functions:

• Counterbalances the effect of glutathione (GSH) depletion on mitochondrial thiol redox potential

• Prevents mitochondrial morphology aberrations induced by oxidative stress

• Regulates mitochondrial thiol/disulfide redox status

• Interacts with mitochondrial monothiol glutaredoxin GLRX5

Research has shown that BOLA1 knockdown causes an oxidative shift in mitochondrial thiol redox potential, confirming its role in maintaining redox homeostasis . Importantly, BOLA1 orthologs only occur in aerobic eukaryotes, suggesting an evolutionary adaptation to oxygen-rich environments .

What applications is BOLA1 antibody suitable for?

BOLA1 antibody (such as 18017-1-AP) has been validated for multiple experimental applications, each with specific recommended dilutions:

It is recommended that researchers titrate the antibody in their specific testing system to obtain optimal results, as the ideal concentration may vary depending on sample type and experimental conditions .

What is the recommended protocol for using BOLA1 antibody in Western blotting?

For Western blotting applications using BOLA1 antibody, follow this methodological approach:

• Sample preparation: Prepare protein lysates from your cells or tissues of interest. BOLA1 antibody has been validated in HEK-293 and HepG2 cells .

• Protein separation: Use standard SDS-PAGE techniques to separate proteins. Since BOLA1 has an observed molecular weight of 14 kDa, adjust your gel percentage accordingly (12-15% is typically suitable).

• Transfer: Transfer proteins to a nitrocellulose or PVDF membrane using standard transfer protocols.

• Blocking: Block the membrane with appropriate blocking buffer (typically 5% non-fat dry milk or BSA in TBST).

• Primary antibody incubation: Dilute BOLA1 antibody in blocking buffer at a ratio of 1:500 to 1:2000 . Incubate the membrane with this solution overnight at 4°C or for 1-2 hours at room temperature.

• Washing: Wash the membrane 3-5 times with TBST.

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG for 18017-1-AP).

• Detection: Use enhanced chemiluminescence (ECL) reagents for detection.

The antibody should detect a band at approximately 14 kDa, corresponding to the BOLA1 protein . Always include positive controls such as HEK-293 or HepG2 cell lysates where BOLA1 expression has been validated.

What cell types or tissues show high expression of BOLA1?

BOLA1 expression has been detected in various cell types and tissues. Based on immunohistochemistry data, BOLA1 antibody has been validated for detection in:

• Human lung tissue

• Human heart tissue

• Human kidney tissue

• Human spleen tissue

For cell culture models, BOLA1 has been successfully detected in:

• HEK-293 cells

• HepG2 cells

As a mitochondrial protein, BOLA1 would be expected to be present in cells with high mitochondrial content, such as muscle cells, hepatocytes, and neurons. Its presence across multiple tissues suggests that it plays a fundamental role in mitochondrial function across different cell types, particularly in managing redox homeostasis in aerobic conditions .

How should I validate the specificity of BOLA1 antibody in my experiments?

To ensure the specificity of BOLA1 antibody in your experiments, implement these validation strategies:

• Positive controls: Use cell lines with known BOLA1 expression such as HEK-293 or HepG2 cells .

• BOLA1 knockdown: Employ siRNA against BOLA1 mRNA to knockdown expression. The research literature identifies three effective siRNAs for BOLA1 knockdown:

  • #1 antisense strand: 5′-UUAACAUGGAACAUCCGGGdTdT

  • #2 antisense strand: 5′-UUGUUUCCAACUCAUCAGGdTdT

  • #3 antisense strand: 5′-ACUGUAUCAAAGGGAAGGCdTdT

  After knockdown, perform Western blotting to confirm reduced signal with the BOLA1 antibody.

• BOLA1 overexpression: Overexpress BOLA1-GFP or BOLA1-TAP and confirm increased signal detection.

• Molecular weight verification: Ensure that the detected band appears at the expected molecular weight of 14 kDa .

• Multiple applications: Validate the antibody in different applications (WB, IF, IHC) to ensure consistent results across methodologies.

Implementing multiple validation approaches increases confidence in antibody specificity and experimental reliability. This is particularly important when studying proteins like BOLA1 that may have homologs (such as BOLA2 and BOLA3) with potentially overlapping functions .

What are the best experimental conditions for studying BOLA1's interaction with GLRX5?

The interaction between BOLA1 and GLRX5 (mitochondrial monothiol glutaredoxin) can be studied using several experimental approaches:

• Tandem Affinity Purification (TAP):

  • Generate cells that inducibly express BOLA1 or GLRX5 with a C-terminal TAP tag

  • After induction (24 hours), prepare cell lysates for affinity purification

  • Identify interacting proteins by nanospray ionization liquid chromatography tandem mass spectrometry (nLC-MS/MS)

  • Research has shown that when using BOLA1-TAP as bait, GLRX5 was among the specifically copurified mitochondrial proteins

• Co-immunoprecipitation and Western blotting:

  • Use BOLA1-TAP or GLRX5-TAP as bait

  • Verify interaction by Western blotting using antibodies against the potential interacting partner

  • Research demonstrated that BOLA1 was successfully copurified with GLRX5-TAP

• Mixed lysate approach:

  • Mix lysates of BOLA1-TAP-expressing cells with those expressing GLRX5-GFP or GFP alone

  • Incubate with streptactin beads

  • Verify copurification of GLRX5-GFP by Western blotting using anti-GFP

  • This approach resulted in significant BOLA1-TAP copurified GLRX5-GFP compared to GFP control

When studying this interaction, consider that it may be weak or transient, as purification of BOLA1-TAP did not yield detectable amounts of copurified GLRX5 in direct approaches, while the mixed lysate approach was successful . This suggests optimization of buffer conditions or crosslinking strategies may be necessary to capture this interaction effectively.

How can I investigate the role of BOLA1 in mitochondrial redox homeostasis?

To investigate BOLA1's role in mitochondrial redox homeostasis, employ these methodological approaches:

• BOLA1 knockdown studies:

  • Transfect cells with siRNAs against BOLA1 mRNA (see section 1.5 for sequences)

  • Use Dharmafect 1 transfection reagent with Optimem medium

  • Perform two rounds of transfection 48 hours apart for effective knockdown

  • Confirm knockdown efficiency by immunoblot analysis using anti-BOLA1 antibody

• Redox potential measurement:

  • Express redox-sensitive fluorescent proteins such as mito-roGFP1 (mitochondrial) or cyto-roGFP1 (cytosolic)

  • Use digital-imaging microscopy to measure fluorescence indicating thiol/disulfide redox status

  • Compare redox potential between BOLA1 knockdown cells and controls

  • Research demonstrated that BOLA1 knockdown caused an oxidative shift of mitochondrial thiol/disulfide redox status

• Oxidative stress induction:

  • Treat cells with L-buthionine-(S,R)-sulfoximine (BSO) to deplete glutathione

  • Alternatively, use S-nitrosocysteine (SNOC) to induce nitrosative stress

  • Compare effects in BOLA1-overexpressing vs. control cells

  • Research showed that BOLA1 overexpression nullified the effect of BSO and SNOC on mitochondrial morphology

• Reactive oxygen species measurement:

  • Load cells with hydroethidine (HEt) to measure superoxide production

  • Use digital-imaging microscopy to quantify HEt oxidation

  • Compare between BOLA1 knockdown/overexpression and control cells

  • Note that research demonstrated HEt oxidation (superoxide production) and thiol oxidation can be independent processes

These complementary approaches provide a comprehensive understanding of BOLA1's role in maintaining mitochondrial redox homeostasis under normal and stressed conditions.

How does BOLA1 deficiency affect mitochondrial morphology and function?

BOLA1 deficiency has significant effects on mitochondrial morphology and function, particularly related to redox homeostasis:

Effects on Mitochondrial Morphology:
Research has shown that while BOLA1 overexpression itself does not significantly alter normal mitochondrial morphology parameters, BOLA1 plays a critical role in preventing morphological changes induced by oxidative stress:

• Under glutathione depletion (BSO treatment):

  • Normal mitochondria become shorter and less branched

  • BOLA1 overexpression completely prevents these BSO-induced morphological alterations

• Key morphological parameters affected:

  • Mitochondrial area

  • Aspect ratio (AR, a measure of mitochondrial length)

  • Form factor (F, a measure of mitochondrial length and degree of branching)

• Important distinction: BSO treatment did not alter the number of mitochondria per cell, suggesting mitochondrial shrinkage rather than fragmentation occurs in BOLA1-deficient conditions

Effects on Mitochondrial Function:

• Redox homeostasis:

  • BOLA1 knockdown causes an oxidative shift in the mitochondrial thiol/disulfide redox status

  • This is measurable using redox-sensitive fluorescent proteins like mito-roGFP1

• Response to stress:

  • BOLA1 deficiency makes mitochondria more vulnerable to oxidative stress from glutathione depletion

  • It also increases susceptibility to nitrosative stress (e.g., from S-nitrosocysteine)

These findings highlight BOLA1's role as a protective factor for maintaining mitochondrial morphology and redox homeostasis under stress conditions, which has potential implications for understanding mitochondrial dysfunction in various pathological states.

What techniques can be used to measure the effect of BOLA1 on thiol redox potential?

Several sophisticated techniques can be employed to measure the effect of BOLA1 on thiol redox potential:

• Redox-sensitive GFP (roGFP) fluorescence:

  • Express mitochondrially-targeted roGFP1 (mito-roGFP1) or cytosolic roGFP1 (cyto-roGFP1)

  • These proteins contain engineered surface-exposed thiols that form reversible disulfide bonds

  • The oxidation state alters the fluorescence properties

  • Use digital-imaging microscopy to measure fluorescence at different excitation wavelengths

  • Calculate the ratio of fluorescence intensities to determine redox state

  • This approach was successfully used to demonstrate that BOLA1 knockdown caused an oxidative shift in mitochondrial thiol/disulfide redox status

• Hydroethidine (HEt) oxidation measurement:

  • Load cells with HEt to measure superoxide production

  • Use digital-imaging microscopy to quantify HEt-specific oxidation products

  • Compare between BOLA1 manipulated cells and controls

  • Research showed that HEt oxidation and thiol oxidation can be independent processes

• NAD(P)H autofluorescence:

  • Measure cellular NAD(P)H levels via autofluorescence

  • Changes in NAD(P)H levels can indicate altered redox metabolism

  • Research noted that BSO treatment significantly increased NAD(P)H levels, and this was not restored by BOLA1 overexpression

These techniques provide complementary information about different aspects of cellular redox homeostasis and how they are affected by BOLA1 expression levels. When designing experiments, consider that each technique measures different aspects of redox biology, and a comprehensive approach using multiple methods will provide the most complete understanding of BOLA1's role.

How can I study the relationship between BOLA1 and oxidative stress response?

To investigate the relationship between BOLA1 and oxidative stress response, implement these methodological approaches:

• Oxidative stress induction models:

  • L-buthionine-(S,R)-sulfoximine (BSO) treatment: Depletes glutathione by inhibiting its synthesis

  • S-nitrosocysteine (SNOC) treatment: Induces nitrosative stress

  • Both have been shown to cause mitochondrial morphology changes that can be prevented by BOLA1 overexpression

• Rescue experiments:

  • Induce oxidative stress in cells with normal, overexpressed, or knocked-down BOLA1

  • Assess whether BOLA1 overexpression can rescue stress-induced phenotypes

  • Compare with other antioxidants like DTT (which mimicked the effect of BOLA1)

  • Research demonstrated that BOLA1 overexpression completely prevented BSO and SNOC-induced changes in mitochondrial shape

• Thiol redox status measurement:

  • Use mito-roGFP1 to measure mitochondrial thiol/disulfide redox status

  • Compare the oxidative shift between control and stressed conditions

  • Determine if BOLA1 manipulation affects this shift

• Target protein identification:

  • Investigate potential target proteins of the BOLA1/GLRX5 complex involved in maintaining normal mitochondrial shape

  • Consider proteins that need to be in a reduced state to perform their function

  • Research suggests that DTT mimicked BOLA1's effect, indicating that a putative target protein needs to be in the reduced state

• Drp1 S-nitrosylation studies:

  • Investigate whether BOLA1 prevents SNOC-induced S-nitrosylation of the mitochondrial fission protein Drp1

  • This is particularly relevant as S-nitrosylated Drp1 has been implicated in neurodegenerative diseases like Alzheimer's

These approaches provide a comprehensive framework for investigating BOLA1's role in oxidative stress response and its protective mechanisms. The research suggests potential clinical relevance, as the protective effects of BOLA1 against S-nitrosylation may have implications for understanding neurodegenerative disease mechanisms.

What are the best cellular models for studying BOLA1 function in relation to mitochondrial dynamics?

When selecting cellular models to study BOLA1 function in relation to mitochondrial dynamics, consider these options and their specific advantages:

• Human cell lines with validated BOLA1 expression:

  • HEK293 cells: Successfully used for BOLA1-TAP and GLRX5-TAP expression studies

  • HepG2 cells: Validated for BOLA1 antibody applications including WB, IP, and IF/ICC

  • HeLa cells: Effectively used for BOLA1 knockdown studies using siRNA

• Fibroblasts:

  • Fibroblasts have been successfully used to study mitochondrial morphology parameters in the context of BOLA1 overexpression

  • They are particularly useful for quantitative analysis of mitochondrial morphology parameters like area, aspect ratio, form factor, and number of mitochondria per cell

  • Their flat morphology makes them ideal for high-resolution imaging of mitochondrial networks

• Mitochondrial visualization techniques:

  • TMRM staining: Used to visualize mitochondria in fibroblasts overexpressing BOLA1-GFP

  • Mito-roGFP1: Used for simultaneous assessment of mitochondrial morphology and redox status

The selection of cellular model should be guided by the specific aspect of BOLA1 function being investigated and the technical requirements of your experimental approach. For studies focusing on mitochondrial dynamics and morphology, fibroblasts offer advantages due to their flat morphology and ease of imaging. For biochemical studies of protein-protein interactions, HEK293 cells have proven effective. For investigating redox effects, all three cell types (HEK293, HepG2, and HeLa) have been successfully employed with appropriate redox-sensitive probes.

How do I reconcile conflicting data regarding BOLA1's role in different experimental systems?

When faced with conflicting data regarding BOLA1's role across different experimental systems, employ these methodological approaches to reconcile discrepancies:

• Comprehensive phenotypic analysis:

  • Investigate multiple aspects of BOLA1 function simultaneously

  • For example, research showed that while BOLA1 overexpression prevented BSO-induced changes in mitochondrial morphology, it failed to restore NAD(P)H levels

  • This indicates that BOLA1 may affect some aspects of cellular response to oxidative stress but not others

• Experimental context consideration:

  • Different stressors: Test multiple stress inducers (e.g., BSO, SNOC) as they may act through different mechanisms

  • Stress intensity: Titrate the level of stress to determine threshold effects of BOLA1

  • Temporal dynamics: Consider time-dependent effects on both stress response and BOLA1 action

• Interaction partners analysis:

  • Investigate whether the expression and activity of interaction partners like GLRX5 varies between experimental systems

  • Research mentioned that BOLA1 knockdown did not alter the amount of GLRX5 , but the activity or post-translational modifications of these partners may still influence BOLA1 function

• Quantitative rather than qualitative analysis:

  • Use precise quantification methods like:

    • Digital-imaging microscopy of roGFP fluorescence for redox potential

    • Quantitative analysis of mitochondrial morphology parameters (area, AR, F, Nc)

  • These approaches allow detection of subtle differences that might explain apparent contradictions

By systematically addressing these factors, researchers can develop a more nuanced understanding of BOLA1's context-dependent functions and reconcile seemingly conflicting experimental results. Remember that biological systems are complex, and proteins often have multiple functions that may be differentially revealed depending on the experimental approach.

What methodological considerations are important when designing experiments to study BOLA1 function?

When designing experiments to study BOLA1 function, consider these critical methodological considerations:

• Expression manipulation strategies:

  • Knockdown: The three validated siRNAs (see section 1.5) have shown different efficiencies; consider testing multiple siRNAs and confirming knockdown by Western blot

  • Overexpression: Tagged versions (GFP, RFP, TAP) of BOLA1 have been successfully used, but consider whether the tag might affect function

  • Rescue experiments: Combine knockdown with re-expression to confirm specificity of observed phenotypes

• Mitochondrial function assessment:

  • When analyzing mitochondrial morphology, distinguish between fragmentation (increased mitochondrial number) and shrinkage (decreased size without number change)

  • Research showed BSO treatment caused mitochondrial shrinkage rather than fragmentation

  • Include multiple parameters: area, aspect ratio, form factor, and number of mitochondria per cell

• Redox status measurement:

  • Consider that different redox parameters may respond differently

  • Research showed HEt oxidation (superoxide) and thiol oxidation can be independent

  • Include measurements of both general and specific redox parameters

  • Combine with functional readouts (e.g., mitochondrial morphology, respiration)

• Interaction studies:

  • The BOLA1-GLRX5 interaction may be weak or transient

  • Direct co-IP failed to detect interaction in one direction, while mixed lysate approach was successful

  • Consider stabilizing interactions with crosslinking or optimized buffer conditions

• Controls for stress experiments:

  • Include antioxidant controls (e.g., DTT) to determine if phenotypes are redox-dependent

  • Research showed DTT mimicked BOLA1's protective effect

  • Include titration of stressors to determine dose-response relationships

By carefully considering these methodological aspects, researchers can design robust experiments that provide reliable and interpretable results about BOLA1 function in mitochondrial redox regulation and morphology maintenance.