HMBS Antibody

What is HMBS and why is it significant in biomedical research?

HMBS (Hydroxymethylbilane Synthase) is a protein-coding gene that encodes the third enzyme in the heme biosynthetic pathway. The enzyme catalyzes the sequential polymerization of four porphobilinogen molecules to form hydroxymethylbilane, also known as preuroporphyrinogen . This process begins with the assembly of the dipyrromethane cofactor from two molecules of porphobilinogen, which then acts as a primer around which the tetrapyrrole product is assembled .

The significance of HMBS in research stems from its critical role in heme metabolism and its association with acute intermittent porphyria, an autosomal dominant disease characterized by neurological dysfunctions, hypertension, abdominal pain, and excessive amounts of aminolevulinic acid and porphobilinogen in the urine . Studying HMBS using antibody-based techniques enables researchers to:

• Investigate heme biosynthesis pathway regulation

• Explore mechanisms underlying porphyria-related disorders

• Understand metabolic adaptations in various physiological and pathological conditions

• Develop potential therapeutic strategies for porphyria

What types of HMBS antibodies are available for research applications?

Researchers can choose from several types of HMBS antibodies optimized for different experimental applications:

Polyclonal antibodies:

• Recognize multiple epitopes on the HMBS protein

• Examples include Thermo Fisher Scientific's PA5-62230 and Aviva Systems Biology's OACD04224

• Generally offer higher sensitivity but may have increased background

Monoclonal antibodies:

• Recognize specific epitopes with high specificity

• Include products like Proteintech's 14797-1-AP

• Provide consistent results across experiments with lower batch-to-batch variation

Available HMBS antibodies have been validated for multiple applications including:

When selecting an HMBS antibody, researchers should consider the specific application requirements, species reactivity, and the region of HMBS targeted by the antibody .

What is the molecular information for HMBS and how does it help in antibody selection?

Understanding the molecular characteristics of HMBS is crucial for appropriate antibody selection and experimental design:

Key molecular characteristics:

• Full name: Hydroxymethylbilane synthase

• Calculated molecular weight: 39 kDa

• Observed molecular weight on gels: 39-42 kDa

• Gene ID (NCBI): 3145

• UniProtKB/Swiss-Prot: P08397

Protein aliases to be aware of when searching literature:

• Porphobilinogen deaminase (PBGD)

• PBG-D

• UPS

• PORC

• Pre-uroporphyrinogen synthase

Previous HGNC symbols for HMBS gene:

• PBGD

• UPS

• PORC

This information helps researchers:

• Verify antibody specificity by confirming the observed molecular weight matches expectations

• Cross-reference between databases when searching for information

• Understand the various nomenclature used in scientific literature

• Identify the specific isoforms or regions targeted by different antibodies

What are the optimal protocols for Western blotting using HMBS antibodies?

For successful Western blot detection of HMBS, consider the following methodological approach:

Sample preparation:

• Extract proteins using standard lysis buffers (RIPA or NP-40 based buffers)

• Include protease inhibitors to prevent degradation

• Determine protein concentration using BCA or Bradford assay

• Load 10-30 μg total protein per lane

Electrophoresis and transfer:

• Use 10-12% SDS-PAGE gels (HMBS is ~39-42 kDa)

• Transfer to PVDF or nitrocellulose membrane

• Verify transfer efficiency with reversible staining (Ponceau S)

Antibody incubation and detection:

• Block with 5% non-fat milk or BSA in TBST

• Incubate with anti-HMBS antibody:

  • Proteintech 14797-1-AP: 1:500-1:2000 dilution

  • Aviva OACD04224: 1:50-400 dilution

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using chemiluminescence detection system

Validated positive controls:

• HeLa cells, A375 cells, HEK-293 cells, Raji cells

Troubleshooting:

• If multiple bands appear: Increase antibody dilution or try different blocking reagent

• If no signal: Decrease antibody dilution, increase protein loading, or verify sample preparation

• For high background: Extend washing steps and increase detergent concentration

Optimization of antibody concentration is critical - start with the manufacturer's recommended dilution range and adjust based on signal-to-noise ratio.

How should immunofluorescence experiments with HMBS antibodies be designed?

Immunofluorescence (IF) and immunocytochemistry (ICC) with HMBS antibodies require careful optimization:

Cell preparation:

• Culture cells on glass coverslips or chamber slides

• Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 in PBS (5-10 minutes)

• Block with 5-10% normal serum (from secondary antibody species) for 1 hour

Antibody staining:

• Incubate with primary HMBS antibody:

  • Proteintech 14797-1-AP: 1:20-1:200 dilution

  • Aviva OACD04224: 1:50-500 dilution for formalin-fixed cells

• Incubate overnight at 4°C or 1-2 hours at room temperature

• Wash 3-5 times with PBS

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Validated cell lines:

• MCF-7 cells, HeLa cells, HepG2 cells

Expected localization:
HMBS is primarily cytoplasmic, so expect diffuse cytoplasmic staining pattern.

Controls to include:

• Primary antibody omission control

• Isotype control

• Positive control using cells known to express HMBS

• Negative control using cells with HMBS knockdown (if available)

Imaging considerations:

• Capture images using similar exposure settings across samples

• Include scale bars in all images

• Consider Z-stack acquisition for thick samples

• Use appropriate filter sets to minimize bleed-through

What are the best practices for immunoprecipitation with HMBS antibodies?

Immunoprecipitation (IP) allows isolation of HMBS protein complexes for downstream analysis:

Sample preparation:

• Prepare cell lysates in non-denaturing lysis buffer (typically containing 1% NP-40 or Triton X-100)

• Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

• Clear lysates by centrifugation (14,000 × g, 10 minutes, 4°C)

• Optional: Pre-clear with Protein A/G beads to reduce non-specific binding

Immunoprecipitation protocol:

• Add HMBS antibody to cleared lysate:

  • Proteintech 14797-1-AP: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Aviva OACD04224: Follow manufacturer's recommendation

• Incubate overnight at 4°C with gentle rotation

• Add protein A/G beads and incubate 2-4 hours at 4°C

• Wash beads 4-5 times with lysis buffer

• Elute bound proteins with SDS sample buffer or specific elution buffer

• Analyze by Western blotting or mass spectrometry

Validated samples:

• HEK-293 cells have been validated for IP with Proteintech antibody

Critical controls:

• IgG control - use the same amount of isotype-matched IgG

• Input control - load 5-10% of pre-IP lysate

• Supernatant control - analyze unbound fraction to assess IP efficiency

Troubleshooting:

• Low IP efficiency: Increase antibody amount, extend incubation time, optimize lysis conditions

• High background: Increase washing stringency, use more selective lysis buffer

• Non-specific bands: Use crosslinking to reduce antibody chain detection

How can HMBS antibodies be optimized for immunohistochemistry in different tissue types?

Immunohistochemistry (IHC) with HMBS antibodies requires tissue-specific optimization:

Tissue preparation:

• For FFPE tissues:

  • Fix in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Cut 4-5 μm sections

• For frozen tissues:

  • Snap freeze in OCT compound

  • Cut 5-10 μm cryosections

  • Fix in cold acetone or 4% PFA

Antigen retrieval optimization:

• Heat-induced epitope retrieval:

  • Citrate buffer (pH 6.0) - Start with this standard approach

  • EDTA buffer (pH 9.0) - Alternative for some epitopes

  • Optimize heating time (10-30 minutes)

• Enzymatic retrieval (alternative):

  • Proteinase K (5-15 minutes)

  • Trypsin (5-15 minutes)

Staining protocol:

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with serum

• Incubate with HMBS antibody:

  • Aviva OACD04224: 1:10-100 for paraffin sections, 1:50-500 for frozen sections

• Apply detection system (ABC, polymer-based)

• Develop with DAB or other chromogen

• Counterstain with hematoxylin

Tissue-specific considerations:

• Liver: Often shows higher HMBS expression; may require higher antibody dilution

• Brain: May need extended antigen retrieval

• Highly pigmented tissues: Consider using alternative chromogens to distinguish from endogenous pigment

Quantification approaches:

• H-score method (combines intensity and percentage of positive cells)

• Digital image analysis for more objective assessment

• Multiplex IHC for contextual information

How can researchers verify HMBS antibody specificity in their experimental system?

Verifying antibody specificity is critical for reliable research findings. Implement these methodological approaches:

Genetic validation:

• CRISPR knockout validation:

  • Generate HMBS knockout cells

  • Compare antibody reactivity between wild-type and knockout

  • Complete absence of signal in knockout cells confirms specificity

• siRNA/shRNA knockdown:

  • Reduce HMBS expression with targeted siRNA

  • Confirm knockdown by qPCR

  • Observe corresponding reduction in antibody signal

Biochemical validation:

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Use this mixture in parallel with regular antibody

  • Specific signal should be blocked by peptide competition

• Recombinant protein controls:

  • Test antibody against purified recombinant HMBS

  • Verify detection at expected molecular weight

Orthogonal validation:

• Multiple antibodies approach:

  • Use antibodies targeting different HMBS epitopes

  • Consistent results across antibodies suggest specificity

• Correlation with mRNA:

  • Compare protein expression (antibody) with mRNA levels (qPCR)

  • Similar patterns support antibody specificity

Application-specific controls:

• For Western blot:

  • Verify single band at 39-42 kDa

  • Check positive control samples (HeLa, HEK-293 cells)

• For IF/IHC:

  • Confirm expected subcellular localization

  • Include primary antibody omission controls

What approaches can be used to study HMBS in relation to acute intermittent porphyria?

Acute intermittent porphyria (AIP) is directly linked to HMBS mutations, making HMBS antibodies valuable tools for investigating this disorder:

Expression analysis in patient samples:

• Western blot quantification:

  • Compare HMBS protein levels between AIP patients and controls

  • Correlate protein levels with clinical manifestations

  • Analyze expression in different tissues using tissue lysates

• Immunohistochemical assessment:

  • Examine HMBS distribution in liver biopsies

  • Compare cellular and subcellular localization between patients and controls

  • Quantify expression differences using digital pathology

Mutation impact studies:

• In vitro expression systems:

  • Express wild-type and mutant HMBS in cell models

  • Use antibodies to assess protein stability and localization

  • Correlate with enzymatic activity measurements

• Structural consequence assessment:

  • Immunoprecipitate wild-type and mutant HMBS

  • Analyze structural differences using limited proteolysis

  • Examine potential protein-protein interaction changes

Therapeutic development applications:

• Compound screening:

  • Use antibodies to monitor HMBS stabilization by small molecules

  • Develop high-throughput screening assays

  • Validate hits in patient-derived cells

• Gene therapy assessment:

  • Evaluate restoration of HMBS expression after gene delivery

  • Compare exogenous vs. endogenous protein levels

  • Monitor tissue-specific expression

Methodological considerations:

• Include both wild-type and mutant HMBS controls

• Consider using antibodies that specifically detect common HMBS mutations

• Combine with enzymatic activity assays for functional correlation

• Account for tissue-specific expression patterns

How can mass spectrometry be combined with HMBS antibodies for advanced protein analysis?

Integrating antibody-based enrichment with mass spectrometry provides powerful analytical capabilities:

Immuno-mass spectrometry workflow:

• Sample preparation:

  • Digest protein samples with trypsin

  • Add stable isotope-labeled HMBS peptide standards

• Antibody enrichment:

  • Couple anti-HMBS antibodies to magnetic beads

  • Incubate digested samples with antibody-beads

  • Wash to remove non-specific binders

  • Elute bound peptides

• MS analysis:

  • Develop multiple reaction monitoring (MRM) assays for HMBS peptides

  • Monitor multiple transitions per peptide

  • Quantify based on endogenous/labeled peptide ratios

Applications in HMBS research:

• Absolute quantification:

  • Measure precise HMBS concentrations in biological samples

  • Compare levels across different tissues or conditions

  • Establish reference ranges for diagnostic applications

• Post-translational modification analysis:

  • Identify phosphorylation, acetylation, or other modifications

  • Compare modification patterns between normal and disease states

  • Correlate modifications with enzymatic activity

• Protein interaction studies:

  • Immunoprecipitate HMBS complexes

  • Identify interacting partners by MS

  • Validate interactions using orthogonal methods

Technical considerations:

• Select antibodies that don't preferentially bind modified peptides

• Include appropriate controls (e.g., immunoprecipitation from HMBS knockout samples)

• Optimize digestion conditions for complete proteolysis

• Consider immunoprecipitation before or after protein digestion

This approach combines antibody specificity with the analytical power of mass spectrometry, enabling detailed characterization of HMBS protein in complex samples .

What are common technical challenges when working with HMBS antibodies and how can they be addressed?

Researchers may encounter several challenges when working with HMBS antibodies. Here are methodological solutions for common issues:

Western blotting challenges:

Immunostaining challenges:

Immunoprecipitation challenges:

General optimization approaches:

• Titrate antibody concentrations to determine optimal working dilution

• Include positive controls (HeLa, HEK-293 cells) in each experiment

• Run parallel negative controls (primary antibody omission, isotype control)

• Prepare all buffers fresh and maintain consistent incubation times/temperatures

How can researchers design experiments to study HMBS in the context of the heme biosynthesis pathway?

Investigating HMBS within the broader heme biosynthesis pathway requires careful experimental design:

Pathway analysis approaches:

• Multi-protein detection:

  • Design antibody panels targeting multiple heme pathway enzymes

  • Perform Western blots or multiplex immunofluorescence

  • Analyze coordinated expression changes across the pathway

• Enzyme activity correlation:

  • Measure HMBS protein levels using antibody-based methods

  • Perform parallel enzyme activity assays

  • Correlate protein expression with enzymatic function

• Regulatory mechanism investigation:

  • Use HMBS antibodies to identify transcription factor binding via ChIP

  • Study post-translational modifications affecting enzyme activity

  • Examine protein-protein interactions through co-immunoprecipitation

Experimental models:

• Cell culture systems:

  • Erythroid cell lines (K562, MEL) with high heme synthesis

  • Hepatocytes with regulated HMBS expression

  • Genetic manipulation models (CRISPR, RNAi)

• Tissue analysis:

  • Liver and bone marrow (major heme synthesis sites)

  • Compare expression across tissues with immunohistochemistry

  • Analyze pathway coordination in disease states

Methodological considerations:

• Pathway perturbation strategies:

  • Heme precursor supplementation or depletion

  • Hypoxia or oxidative stress induction

  • Drug treatments affecting heme metabolism

• Temporal dynamics:

  • Time-course experiments after pathway stimulation

  • Pulse-chase studies to assess protein turnover

  • Circadian rhythm analysis of pathway components

• Control selections:

  • Use housekeeping proteins unaffected by heme metabolism

  • Include both positive regulation and negative feedback controls

  • Consider tissue-specific expression patterns

These approaches enable comprehensive analysis of HMBS within its biological context, providing insights into pathway regulation and disease mechanisms.

How should HMBS antibodies be validated for use in multiplexed immunoassays?

Multiplexed immunoassays require rigorous validation to ensure reliable detection of multiple targets simultaneously:

Pre-multiplexing validation:

• Single-target optimization:

  • Validate each antibody individually before multiplexing

  • Determine optimal working concentrations

  • Confirm specificity using appropriate controls

• Cross-reactivity assessment:

  • Test each antibody against all targets in the panel

  • Perform sequential staining to identify potential interference

  • Conduct antibody omission controls for each component

• Spectral compatibility verification:

  • For fluorescence-based assays, assess fluorophore spectral overlap

  • Create single-color controls for each fluorophore

  • Test various fluorophore combinations to minimize bleed-through

Multiplexing strategies for HMBS studies:

Advanced validation approaches:

• Orthogonal confirmation:

  • Verify multiplexed results with single-plex assays

  • Compare results from different methodological platforms

  • Correlate with gene expression or other orthogonal measures

• Reference standards:

  • Include calibrated reference samples in each run

  • Use samples with known target concentrations

  • Establish internal control metrics for assay performance

• Statistical validation:

  • Assess intra-assay and inter-assay variability

  • Determine limits of detection for each target

  • Validate dynamic range in multiplexed format

These validation approaches ensure that multiplexed assays involving HMBS antibodies provide reliable and reproducible results across experiments .

How can nanobody technology be applied to HMBS research?

Nanobodies (single-domain antibodies) represent an emerging technology with significant potential for HMBS research:

Advantages of nanobodies over conventional antibodies:

• Small size (~15 kDa vs. ~150 kDa for IgG)

• High stability and solubility

• Efficient tissue penetration

• Access to cryptic epitopes

• Cost-effective production

• Simple engineering into multivalent formats

Applications in HMBS research:

• Live-cell imaging:

  • Generate fluorescently tagged anti-HMBS nanobodies

  • Track HMBS dynamics in living cells

  • Study real-time changes in localization and interactions

• Super-resolution microscopy:

  • Use nanobodies for improved resolution due to reduced linkage error

  • Achieve precise localization of HMBS in subcellular compartments

  • Perform multi-color super-resolution with orthogonal nanobodies

• Intracellular targeting:

  • Express intrabodies targeting HMBS in living cells

  • Disrupt specific interactions or functions

  • Create targeted protein degradation systems

• Therapeutic exploration:

  • Develop nanobodies targeting mutant HMBS forms

  • Create delivery systems for enzyme replacement

  • Design diagnostics for porphyria-related disorders

Methodological approaches:

• Nanobody development:

  • Immunize camelids with purified HMBS protein

  • Construct phage display libraries from B cells

  • Select specific binders through panning

  • Characterize binding properties and epitopes

• Engineering strategies:

  • Create bispecific constructs targeting HMBS and interacting proteins

  • Develop nanobody-based biosensors for HMBS activity

  • Fuse with cell-penetrating peptides for intracellular delivery

The evolutionary innovation of heavy-chain antibodies in camelids has enabled this technology, which offers significant advantages for certain research and therapeutic applications .

What are emerging methods for developing and validating antibodies for rare proteins like HMBS?

Advanced technologies are transforming antibody development and validation processes:

Next-generation antibody development approaches:

• Recombinant antibody technologies:

  • Phage display libraries for antibody selection

  • Yeast display for affinity maturation

  • Mammalian display for functional screening

  • Selection under defined conditions for application-specific antibodies

• In silico design and screening:

  • Computational prediction of antibody structures

  • Virtual screening of antibody-antigen interactions

  • Knowledge-based approaches like AbPredict algorithm

  • Machine learning for epitope prediction

• Synthetic antibody libraries:

  • Rationally designed frameworks with diverse CDRs

  • Scaffold-based binding proteins

  • Minimalist antibody designs

High-throughput validation methodologies:

• Automated screening platforms:

  • Parallel testing of multiple antibody candidates

  • Robotics-based sample handling and analysis

  • High-content imaging for cellular assays

• Developability assessment:

  • Early-stage evaluation of antibody properties

  • Predicting stability, solubility, and manufacturability

  • High-throughput biophysical characterization

• Integrated validation workflows:

  • Combining multiple testing platforms

  • Standardized protocols across applications

  • Comprehensive data management systems

Example integrated workflow from discovery to validation:

These advances in antibody technology enable the development of higher quality reagents for challenging targets like HMBS, supporting more reliable and reproducible research outcomes .