cyb5r4 Antibody

What is CYB5R4 and what biological functions make it a significant research target?

Cytochrome B5 Reductase 4 (CYB5R4), also known as b5+b5R or Ncb5or, is a highly conserved NAD(P)H flavoheme reductase that contains a cytochrome b5-like domain at the N-terminus and a cytochrome b5 reductase-like domain at the C terminus. It is localized in the endoplasmic reticulum (ER) and is ubiquitously expressed, with relatively high mRNA levels in the pancreas, heart, and kidney .

CYB5R4 serves crucial metabolic functions:

• Provides an alternative source of electrons for fatty acid desaturation

• Protects cells against endoplasmic reticulum stress

• Regulates phosphoprotein phosphatase activities (particularly PP4 and PP6)

• Functions in lipid metabolism, which affects healthspan and aging

Knockout studies in mice demonstrate that CYB5R4 deficiency leads to glucose intolerance at approximately 4 weeks of age, followed by development of frank diabetes at 6 weeks. This is accompanied by progressive loss of pancreatic β-cells and white adipose tissue, highlighting its importance in metabolic regulation .

What applications are CYB5R4 antibodies validated for in research protocols?

CYB5R4 antibodies have been validated for multiple applications, with successful use dependent on proper optimization. Based on available data, these applications include:

The expected molecular weight for CYB5R4 detection is approximately 56 kDa , which serves as a critical validation point when confirming antibody specificity.

What is the recommended experimental design for proper CYB5R4 antibody validation?

A robust validation strategy for CYB5R4 antibodies should include:

• Positive control selection: Use tissues/cells known to express CYB5R4, such as BxPC-3 cells, HeLa cells, or C6 cells . Pancreatic tissue is particularly valuable given CYB5R4's high expression pattern.

• Negative controls:

  • Isotype controls to identify non-specific binding

  • Peptide blocking/competition assays with the immunizing peptide

  • When possible, tissues/cells from CYB5R4 knockout models as the gold standard control

• Cross-reactivity testing: Verify antibody performance across relevant species (human, mouse, rat are commonly validated) .

• Validation across multiple techniques: Confirm CYB5R4 detection using at least two independent methods (e.g., WB plus IHC or IF).

• Specificity verification: Use CYB5R4 knockout/knockdown systems to confirm signal loss. As noted in recent literature, "CYB5R4 knockout was verified by Western blot analysis" .

How should researchers interpret fluorescence-based detection systems for CYB5R4?

When using fluorescence-based detection systems (particularly with conjugated antibodies like AbBy Fluor® 555), consider:

• Autofluorescence mitigation: Tissues with high flavoprotein content may exhibit background autofluorescence that overlaps with some fluorophores. Include unlabeled controls to establish baseline fluorescence.

• Signal-to-noise optimization: For AbBy Fluor® 555 conjugated antibodies, use appropriate filter sets (excitation maximum ~555 nm, emission maximum ~565 nm) .

• Data normalization: When conducting quantitative fluorescence measurements, use the procedure described by Eckel et al. whereby "intensity-dependent biases" are corrected through nonparametric local regression procedures .

• Multiplexing considerations: When co-staining with other antibodies, select fluorophores with minimal spectral overlap and perform proper compensation/controls.

• Photobleaching prevention: Minimize exposure to excitation light and use antifade mounting media when performing immunofluorescence microscopy.

How can researchers effectively use CYB5R4 antibodies to investigate its role in fatty acid metabolism?

To investigate CYB5R4's role in fatty acid metabolism:

• Co-localization studies: Use dual immunofluorescence with CYB5R4 antibodies and markers for fatty acid desaturation enzymes (such as SCD1). This reveals potential functional relationships, as studies have shown that CYB5R4 provides an alternative source of electrons for fatty acid desaturation .

• Metabolic labeling experiments: Combine CYB5R4 immunoprecipitation with radioactive or stable isotope-labeled fatty acid precursors to track metabolic flux through desaturation pathways.

• Lipidomic profiling: Compare lipid profiles between normal and CYB5R4-depleted samples. Evidence shows that increased expression of CYB5R3 (a related protein) was associated with "accumulation of high levels of long-chain polyunsaturated fatty acids" .

• Fatty acid challenge experiments: Expose cells to palmitic acid (saturated) versus oleic acid (unsaturated) treatments with subsequent CYB5R4 immunoblotting to assess protein expression changes .

• Interactome analysis: Use CYB5R4 antibodies for co-immunoprecipitation followed by mass spectrometry to identify interaction partners involved in fatty acid metabolism.

What is the significance of heme-coordinating residues in CYB5R4 and how does this impact antibody selection?

Recent research has revealed the critical importance of heme-coordinating residues in CYB5R4 function:

• Structural significance: Base editor screening revealed that heme-coordinating histidines (H89/H112) and a proximal tryptophan residue (W114) are essential for CYB5R4 function . Mutations in these residues resulted in hypersensitivity to okadaic acid and cisplatin.

• Protein interaction impacts: When H89/H112 or W114 were mutated to alanine, binding of PP4 and PP6 components to CYB5R41-153 was lost , indicating these residues are critical for protein-protein interactions.

• Antibody selection implications:

  • Antibodies targeting regions containing these critical residues may exhibit altered binding in samples with mutations

  • For studies involving mutant forms of CYB5R4, select antibodies targeting conserved epitopes distant from these heme-coordinating residues

  • When studying the Cytb5 domain specifically, consider antibodies targeting the N-terminal region (AA 21-120)

• Functional domain considerations: The Cytb5 domain was found to be necessary and sufficient for rescuing sensitivity to okadaic acid , suggesting this domain should be prioritized when selecting antibodies for functional studies.

How can CYB5R4 antibodies be optimized for studying its role in phosphatase regulation?

Recent groundbreaking research has revealed that CYB5R4 functions as an activator of PP4 and PP6 phosphatases . To effectively study this regulatory role:

• Co-immunoprecipitation optimization:

  • Use antibodies targeting the Cytb5 domain (amino acids 1-153), as this region is sufficient for PP4/PP6 interaction

  • Perform IPs under native conditions that preserve protein-protein interactions

  • Include appropriate phosphatase inhibitors to preserve phosphorylation states

• Phosphatase activity assays:

  • Couple CYB5R4 immunoprecipitation with phosphatase activity measurements

  • Include controls with heme-coordinating residue mutants (H89A/H112A or W114A) which abolish PP4/PP6 binding

• DNA damage response studies:

  • Use anti-CYB5R4 antibodies in combination with phospho-specific antibodies for PP4 substrates

  • CYB5R4, PP4C, and PPP6C clustered together in DNA damaging agent responses, suggesting "mechanisms maintaining normal PP4 activity is key in determining response to DNA damaging agents"

• Proximity ligation assays (PLA):

  • Utilize CYB5R4 antibodies paired with PP4/PP6 antibodies to visualize in situ interactions

  • This technique can validate AlphaFold 3 predictions about interaction between the Cytb5 domain and phosphatase catalytic subunits

What methodological approaches are recommended for investigating the relationship between CYB5R4 and diabetes?

To effectively investigate CYB5R4's role in diabetes:

• Pancreatic β-cell studies:

  • Optimize immunohistochemistry protocols for pancreatic tissue following fixation in "either buffered 4% paraformaldehyde solution or Bouin's solution"

  • Use co-staining with insulin antibodies to assess β-cell function in relation to CYB5R4 expression

  • CYB5R4 knockout mice show reduced insulin content and progressive loss of β-cells in pancreatic islets

• Genetic association studies:

  • Examine SNPs in CYB5R4 in context of diabetes risk - while limited evidence exists for NCB5OR variants associating directly with type 2 diabetes, mature-onset diabetes of the young, or gestational diabetes, further investigation is warranted

• ER stress investigations:

  • Use CYB5R4 antibodies alongside markers of ER stress (BiP/GRP78, XBP-1, CHOP, ATF3)

  • Examine how CYB5R4 expression levels correlate with ER stress markers under various metabolic challenges

• Human sample analysis:

  • Employ CYB5R4 antibodies in tissue microarrays comparing diabetic and non-diabetic pancreatic samples

  • Quantify expression differences using standardized immunohistochemistry scoring methods

• Metabolic pathway analysis:

  • Combine CYB5R4 antibody-based detection with metabolomic profiling to correlate protein expression with specific metabolic alterations in diabetic models

How can researchers effectively troubleshoot inconsistent results when using CYB5R4 antibodies?

When encountering inconsistent results with CYB5R4 antibodies, implement this systematic troubleshooting approach:

• Antibody validation verification:

  • Confirm antibody detection of endogenous levels using positive control samples (BxPC-3, HeLa, or C6 cells)

  • Verify expected molecular weight detection (~56 kDa)

• Protocol optimization:

  • For Western blotting, test different extraction buffers as CYB5R4 is membrane-associated

  • For IHC, compare antigen retrieval methods (TE buffer pH 9.0 versus citrate buffer pH 6.0)

  • Adjust antibody concentration based on signal strength (1:500-1:2000 for WB; 1:50-1:500 for IHC)

• Sample preparation considerations:

  • Ensure protein integrity through proper sample handling

  • For tissue samples, optimize fixation conditions as different fixatives may affect epitope accessibility

• Species-specific validation:

  • Verify antibody cross-reactivity with your species of interest

  • Some antibodies show restricted species reactivity while others demonstrate broad cross-reactivity

• Experimental design adjustments:

  • Implement the balanced block design described by Kerr et al. for two-color systems to account for potential dye-specific effects

  • For fluorescence-based detection, apply normalization methods to correct for intensity-dependent biases

What are the latest methodological advances in studying CYB5R4 using CRISPR-based approaches?

Recent innovations in CRISPR technology offer powerful approaches to study CYB5R4 function:

• Base editing technology applications:

  • Base editor tiling screens have provided "a high-resolution map of phosphatase function and regulation"

  • This approach enabled identification of critical residues like the heme-coordinating histidines (H89/H112) and tryptophan (W114)

• CRISPR knockout validation:

  • CYB5R4 knockout cell lines serve as essential negative controls for antibody validation

  • As documented in recent research: "CYB5R4 knockout was verified by Western blot analysis"

• Functional domain mapping:

  • CRISPR-based mutagenesis of specific domains (Cytb5, CS, Cytb5-R) can be paired with antibody detection to assess domain-specific functions

  • The Cytb5 domain alone (amino acids 1-153) was found sufficient for PP4/PP6 interaction

• Rescue experiments:

  • After CRISPR knockout, reintroduction of wild-type or mutant CYB5R4 variants can be monitored with domain-specific antibodies

  • These experiments revealed that "the Cytb5 domain was necessary and sufficient for rescuing the sensitivity to OA [okadaic acid]"

• CRISPResso2 mutational analysis:

  • Deep sequencing confirmation of CRISPR edits ensures precise genetic manipulation

  • This approach verified "efficient introduction of the predicted mutations targeting H89, H112, and W114"

What are the key considerations for accurately quantifying CYB5R4 levels in complex biological samples?

For precise quantification of CYB5R4:

• ELISA optimization:

  • When using human CYB5R4 ELISA kits, the validated detection range is 0.156 ng/ml - 10 ng/ml with sensitivity < 0.094 ng/ml

  • Sample types include tissue homogenates, cell lysates, and other biological fluids

• Western blot quantification:

  • Use loading controls appropriate for your sample type (β-actin is commonly used)

  • Implement densitometry with linear range validation

  • For membrane proteins like CYB5R4, compare different extraction methods to ensure complete solubilization

• Adjustment for sample-specific variables:

  • Control for post-translational modifications that may affect antibody binding

  • Consider potential splice variants when interpreting band patterns

• Reference standard inclusion:

  • When possible, include recombinant CYB5R4 protein as a standard curve

  • For relative quantification, establish appropriate reference samples

• Statistical analysis:

  • Apply appropriate statistical tests for comparing CYB5R4 levels between groups

  • As noted in published protocols: "Statistical significance was calculated by the Student's t-test"

What immunoprecipitation strategies work best for studying CYB5R4 protein complexes?

Optimized immunoprecipitation (IP) strategies for CYB5R4:

• Antibody selection criteria:

  • Choose antibodies purified by methods that minimize co-eluting proteins

  • Some CYB5R4 antibodies are "purified by peptide affinity chromatography using SulfoLink Coupling Resin" or "Protein A"

  • N-terminal targeting antibodies effectively capture intact CYB5R4 complexes

• Lysis buffer optimization:

  • Use mild, non-denaturing buffers that preserve protein-protein interactions

  • Include appropriate phosphatase inhibitors when studying CYB5R4-phosphatase complexes

• Specialized techniques:

  • For transient interactions, consider crosslinking prior to lysis

  • Sequential IPs can be used to isolate specific subcomplexes

• Analysis of co-precipitating proteins:

  • Mass spectrometry of immunoprecipitates has revealed CYB5R4 interactions with PP4 and PP6 components

  • Western blotting can confirm specific interactions with known partners

• Controls:

  • Include IgG-matched negative controls

  • CYB5R4 knockout lysates provide stringent negative controls

  • Peptide competition can confirm antibody specificity in IP applications

How can researchers effectively use CYB5R4 antibodies in studying its role in oxidative stress pathways?

To investigate CYB5R4's role in oxidative stress:

• Co-localization with redox markers:

  • Use immunofluorescence to examine CYB5R4 distribution relative to mitochondria and other ROS-generating organelles

  • CYB5R1 (a related protein) has been proposed to localize to mitochondria, "where the enzyme likely plays a pivotal role in oxidative phosphorylation and ROS generation"

• Oxidative challenge studies:

  • Monitor CYB5R4 expression changes during oxidative stress using quantitative immunoblotting

  • CYB5R proteins are required for "elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders"

• Redox proteomics integration:

  • Combine CYB5R4 antibody-based detection with analysis of protein oxidation states

  • Consider the interaction between CYB5R proteins and their role in protecting against oxidative damage

• Ferroptosis pathway analysis:

  • While primarily studied for CYB5R1, investigate whether CYB5R4 has a similar role in ferroptosis regulation

  • "The link between CYB5R1 and ferroptosis could also be important in various cell types, especially primary cells, and outside the realm of cancer research"

• Transgenic model systems:

  • Study the effects of CYB5R overexpression using appropriate antibody detection methods

  • "CYB5R-overexpressing flies (CYB5R-OE) and... transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg)" showed improvements in metabolic parameters and modest lifespan extension

What are the best practices for using CYB5R4 antibodies in examining tissue-specific expression patterns?

For optimal tissue expression profiling:

• Multi-tissue panel analysis:

  • Systematically examine CYB5R4 expression across tissues using standardized IHC protocols

  • Pay particular attention to tissues with reported high expression: pancreas, heart, and kidney

• Cell-type specific resolution:

  • Combine CYB5R4 antibodies with cell-type markers for co-localization studies

  • This approach can reveal differential expression within complex tissues

• Antigen retrieval optimization:

  • Compare multiple antigen retrieval methods for each tissue type

  • For CYB5R4, both "TE buffer pH 9.0" and "citrate buffer pH 6.0" have been effective

• Expression quantification:

  • Develop standardized scoring systems for comparative analysis

  • Consider digital pathology approaches for unbiased quantification

• Tissue microarray (TMA) application:

  • Use TMAs for high-throughput screening across multiple tissue types

  • This approach enables efficient optimization of antibody conditions before proceeding to full tissue sections

How can researchers integrate CYB5R4 antibody data with phosphoproteomic analyses?

To effectively combine CYB5R4 antibody data with phosphoproteomics:

• Experimental design integration:

  • Split samples for parallel antibody-based detection and phosphoproteomic analysis

  • This approach can correlate CYB5R4 expression levels with global phosphorylation changes

• Phosphatase inhibitor beads with mass spectrometry (PIB-MS):

  • This technique has revealed "the specific loss of PP4 and PP6 but not PP2A holoenzyme components in CYB5R4 KO cells"

  • Use CYB5R4 antibodies to confirm knockout efficiency in parallel with PIB-MS

• Kinase-phosphatase network analysis:

  • Map CYB5R4-dependent phosphorylation changes to specific signaling networks

  • Consider the specific effects on "PP4 and PP6 holoenzyme components"

• Functional validation:

  • Use phospho-specific antibodies against known PP4/PP6 substrates to validate mass spectrometry findings

  • This can confirm the functional consequences of CYB5R4's role as an activator of these phosphatases

• Integrative data analysis:

  • Apply computational approaches to integrate antibody-based protein quantification with phosphosite abundance data

  • This can reveal mechanistic insights into how CYB5R4 regulates specific phosphorylation events through its activation of PP4 and PP6