BPGM Antibody

What is BPGM and what biological role does it play?

BPGM (2,3-bisphosphoglycerate mutase) is an enzyme that plays a crucial role in regulating hemoglobin oxygen affinity by controlling levels of its allosteric effector 2,3-bisphosphoglycerate (2,3-BPG). It exhibits mutase (EC 5.4.2.11) activity and serves as an important regulator in the glycolytic pathway . BPGM is particularly significant in contexts requiring oxygen regulation, including erythrocyte function, placental development, and various pathological conditions where oxygen sensing is disrupted, such as cancer and sepsis .

What types of BPGM antibodies are commercially available for research?

Several validated BPGM antibodies are available for research applications, including:

These antibodies have been validated through various methods, including orthogonal RNA sequencing for enhanced validation .

What are the optimal storage conditions for BPGM antibodies?

For most BPGM antibodies, the following storage guidelines apply:

• Store at -20°C for long-term preservation

• Antibodies are typically provided in buffered aqueous glycerol solutions

• Most preparations remain stable for one year after shipment when properly stored

• For the Proteintech 17173-1-AP antibody, aliquoting is unnecessary for -20°C storage

• Some preparations contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

For short-term storage (up to one month), some antibody components may be stored at 4°C, but critical components like the standard, detection reagents, and strip plates should remain at -20°C .

What are the recommended sample types for BPGM detection?

BPGM can be detected in various sample types depending on the experimental goals:

For clinical samples, BPGM has been successfully detected in liver tissue, placental tissue, and plasma from sepsis patients .

How can I optimize BPGM antibody detection for co-localization studies in placental tissue?

For co-detection of BPGM with other markers (such as SynI, SynII, HIF1a, and HIF2a) in placental tissue, consider the following protocol:

• Implement a combined HCR IF + HCR RNA-FISH protocol for FFPE sections (as described by Molecular Instruments; Schwarzkopf et al., 2021)

• Use anti-BPGM antibody at 1:50 dilution

• Perform antigen retrieval with citric acid (pH=6)

• Include probes designed for your co-detection targets

• For imaging, a Dragonfly spinning disc (Andor, Oxford instruments) on a DMi8 microscope (Leica Microsystems) equipped with a Zyla 4.2 camera and a 63× glycerol objective has shown good results

• For quantification, create binned intensity histograms of pixels expressing BPGM signal above a minimal background value (e.g., 1000) in single slices using Fiji Macro

• Important: Since RBCs have high auto-fluorescence in all channels, discard RBC regions prior to BPGM quantification by creating Surface object for RBC in Imaris and setting values in RBC regions to zero

This approach allows for precise spatial correlation between BPGM and other molecular markers in complex placental structures.

What methodological approaches can address epitope masking when using BPGM antibodies in formalin-fixed tissues?

When working with BPGM antibodies in formalin-fixed tissues, epitope masking can be a significant challenge. The following methodological approach has been validated:

• Dewax and rehydrate slides in xylene followed by a series of ethanol washes

• Perform heat-induced epitope retrieval using a pressure cooker with citrate buffer (pH=6)

• Block non-specific binding with 20% NHS and 0.2% Triton in PBS

• For the Sigma-Aldrich HPA016493 antibody, use at 1:200 dilution

• For secondary detection, an HRP anti-Rabbit secondary antibody at 1:100 has proven effective (e.g., Jackson ImmunoResearch Labs, Cat# 111-035-003)

• For fluorescent visualization, Opal 690 at 1:500 dilution has worked well (Akoya Biosciences)

• Always include negative controls incubated with secondary antibody only

• For difficult tissues like placenta with high background, consider using color thresholding in ImageJ for quantification, with multiple measurements (approximately 10) per sample

This approach has successfully been used to detect BPGM in placental tissues where protein expression can be heterogeneous and background signal challenging.

How should BPGM antibody dilution be optimized when comparing expression across different tissue types?

When comparing BPGM expression across different tissue types, antibody dilution optimization is critical to ensure comparable results:

• Perform initial titration experiments using a dilution series (e.g., for IHC: 1:50, 1:100, 1:200, 1:400, 1:800)

• For each tissue type, identify the optimal dilution that provides:

  • Clear specific signal

  • Minimal background

  • Linear range of detection

• Consider tissue-specific recommendations:

  • For liver tissues: HPA016493 at 1:200-1:500 or 17173-1-AP at 1:100-1:400

  • For placental tissues: HPA016493 at 1:50-1:200

  • For cells in culture: ab97497 at 1:100 for IF or 1:1000 for WB

• Standardize protein loading amounts across different sample types

• Include appropriate positive and negative control tissues in each experiment

• For quantitative comparisons, utilize standardized image acquisition parameters and analyze using identical thresholding approaches

It's important to note that different tissue types may require different antigen retrieval methods; for example, TE buffer at pH 9.0 is suggested for liver tissue with the 17173-1-AP antibody, while citrate buffer at pH 6.0 has been effective for placental tissue .

What are the considerations for using BPGM antibodies as prognostic biomarkers in sepsis research?

Recent research has identified BPGM as a potential prognostic marker in sepsis. When designing studies to evaluate BPGM as a biomarker:

• Patient selection criteria should exclude those with pre-existing cardiac diseases that might confound BPGM's relationship with myocardial dysfunction

• Standardize sample collection timing relative to sepsis onset

• Consider correlating BPGM levels with established clinical parameters:

  • APACHE II scores (significantly higher in BPGM-positive patients, 27 vs. 23, p=0.022)

  • Cardiac troponin I levels (significantly higher in BPGM-positive patients, 0.18 vs. 0.04, p=0.033)

  • LVEF (Simpson's method) (significantly lower in BPGM-positive patients, 45% vs. 50%, p<0.01)

  • Tei index (significantly higher in BPGM-positive patients, 0.62 vs. 0.39, p<0.01)

• Establish clear criteria for BPGM positivity

• Track 28-day mortality as a primary outcome (significantly higher in BPGM-positive patients, 54.3% vs. 10.0%, p<0.001)

• Consider combined analysis with other glycolytic pathway markers

This approach has successfully demonstrated that BPGM positivity is associated with poorer heart function and higher mortality in septic patients.

What are the validated protocols for BPGM antibody use in Western blotting?

For optimal Western blot results with BPGM antibodies:

• Sample preparation:

  • Prepare whole cell lysates or tissue homogenates in appropriate lysis buffer

  • Load 30 μg of protein per lane for cell lysates (validated with 293T, A431, and H1299 cells)

• Electrophoresis:

  • Use 12% SDS-PAGE for optimal separation

  • BPGM has a predicted band size of 30 kDa

• Antibody dilutions:

  • Primary antibody:

    • ab97497: 1/1000 dilution

    • 17173-1-AP: 1:1000-1:8000 dilution (sample-dependent)

    • HPA016493: 0.04-0.4 μg/mL

• Detection systems:

  • HRP-conjugated secondary antibodies followed by ECL detection have been validated

  • Fluorescent secondary antibodies may also be used for multiplexing

• Controls:

  • Include positive controls such as HEK-293T cells, human plasma, mouse/rat liver tissue

  • For tissue-specific detection, mouse heart and lung tissues have been validated

Using these parameters has successfully detected BPGM in multiple human cell lines and tissue samples.

How can researchers troubleshoot non-specific binding when using BPGM antibodies in immunohistochemistry?

When encountering non-specific binding in BPGM immunohistochemistry:

• Optimize blocking conditions:

  • Increase blocking duration (try 1-2 hours at room temperature)

  • Test different blocking agents (20% NHS with 0.2% Triton in PBS has been validated)

  • Consider using commercial blocking solutions specific to your tissue type

• Adjust antibody concentrations:

  • For HPA016493: Try more dilute preparations (1:300-1:500)

  • For 17173-1-AP: Start at 1:400 and adjust as needed

• Optimize antigen retrieval:

  • For difficult tissues, compare citrate buffer (pH 6.0) vs. TE buffer (pH 9.0)

  • Standardize retrieval time and temperature

• Address tissue-specific challenges:

  • For tissues with high RBC content (e.g., placenta), account for auto-fluorescence

  • Use appropriate filters or computational methods to subtract auto-fluorescence

• Include validated controls:

  • Always run negative controls (secondary antibody only)

  • Include known positive tissue samples as internal standards

These approaches have been effective in optimizing signal-to-noise ratio in BPGM immunohistochemistry across multiple tissue types.

How is BPGM antibody being used to investigate placental oxygenation during pregnancy?

Recent research has employed BPGM antibodies to investigate placental oxygenation mechanisms:

• Research focus: The role of BPGM enzyme and its product 2,3-BPG in placental oxygenation during pregnancy

• Methodology:

  • BPGM detection in placental labyrinth using immunohistochemistry

  • Co-detection of BPGM with syncytin markers (SynI, SynII) and hypoxia-inducible factors (HIF1a, HIF2a)

  • Quantification of BPGM expression in specific placental compartments:

    • Labyrinth regions containing fetal RBCs

    • Spiral artery trophoblast giant cells (SpA TGCs)

• Analytical approaches:

  • Fractional area expressing both BPGM and containing fetal RBCs measured using color thresholding in ImageJ

  • Spiral arteries diameter measured manually

  • RBC levels in labyrinth assessed via auto-fluorescence signal thresholding

• Findings:

  • BPGM appears to play a role in maternal-fetal oxygen transfer

  • Potential involvement in the pathophysiology of fetal growth restriction (FGR)

This research demonstrates how BPGM antibodies can be leveraged to understand complex physiological processes during pregnancy.

What is the current understanding of BPGM's role in myocardial dysfunction during sepsis?

BPGM antibodies have recently contributed to understanding myocardial dysfunction in sepsis:

• Study design:

  • 85 sepsis patients were categorized into BPGM-positive (n=35) and BPGM-negative (n=50) groups

  • Cardiac function was assessed via echocardiography and serum biomarkers

• Key findings:

  • BPGM-positive patients exhibited:

    • Higher APACHE II scores (27 vs. 23, p=0.022)

    • Elevated cardiac troponin I (0.18 vs. 0.04, p=0.033)

    • Reduced left ventricular ejection fraction (45% vs. 50%, p<0.01)

    • Higher Tei index (0.62 vs. 0.39, p<0.01)

    • Significantly higher 28-day mortality (54.3% vs. 10.0%, p<0.001)

• Mechanistic insights:

  • BPGM likely functions as an intermediate in the glycolytic pathway

  • May influence cardiac function through modulation of 2,3-BPG concentrations

  • Altered glycolytic metabolism in cardiac tissue during sepsis may contribute to myocardial dysfunction

• Clinical implications:

  • BPGM shows potential as a prognostic marker in sepsis

  • May help identify patients at higher risk for cardiac complications and mortality

This research highlights BPGM's emerging role as both a biomarker and potential mechanistic contributor to sepsis-induced cardiac dysfunction.

What are the optimal ELISA conditions for quantitative measurement of BPGM in human samples?

For quantitative measurement of BPGM using ELISA:

• Sample preparation:

  • Human tissue homogenates: Prepare in appropriate extraction buffer (10 mM ammonium acetate/5 mM ammonium bicarbonate, pH 7.7 and methanol in ratio 1:3 by volume)

  • Cell lysates: Extract in compatible lysis buffers that maintain protein integrity

  • Add internal standards (e.g., methionine sulfone at 1 μg/mL) for quantification accuracy

• Protocol parameters:

  • Use sandwich enzyme immunoassay format with pre-coated 96-well plates

  • Ensure all components reach room temperature (18-25°C) before use

  • Dilute samples appropriately to fall within the assay's linear range

  • Include standard curves using the provided reference standard

• Detection system:

  • Use microplate reader with 450 ± 10nm filter

  • For optimal sensitivity, read absorbance within 5 minutes of stop solution addition

• Quality control:

  • Run samples in duplicate or triplicate

  • Include positive and negative controls

  • Calculate intra- and inter-assay coefficients of variation

These conditions have been validated for detecting BPGM in human tissue homogenates, cell lysates, and biological fluids.

How can multiplexed detection approaches be optimized for BPGM co-localization studies?

For multiplexed detection involving BPGM:

• Antibody selection for multiplexing:

  • Choose primary antibodies raised in different host species when possible

  • If using rabbit polyclonal anti-BPGM antibodies, pair with mouse monoclonal antibodies against other targets

  • Confirm antibody performance individually before multiplexing

• Sequential staining approach:

  • Start with the weaker signal (typically BPGM) first

  • Use tyramide signal amplification systems (e.g., Opal 690 at 1:500 dilution)

  • Perform heat-mediated antibody stripping between rounds if using same-species antibodies

• Combined IF and RNA-FISH protocols:

  • The HCR IF + HCR RNA-FISH protocol for FFPE sections has been validated

  • Use anti-BPGM antibody (1:50 dilution) with RNA probes for other targets

  • Ensure complete antibody elution between staining rounds

• Imaging considerations:

  • Use spectral imaging to separate closely overlapping fluorophores

  • Perform sequential acquisition to minimize bleed-through

  • Employ spinning disc confocal microscopy for optimal spatial resolution

• Analysis approaches:

  • Create Surface objects in Imaris for RBC regions to eliminate auto-fluorescence

  • Apply color thresholding in ImageJ for quantification

  • Establish co-localization coefficients using appropriate software

These approaches have successfully demonstrated BPGM co-localization with hypoxia-inducible factors and syncytin markers in placental research.

What genetic testing approaches are available for studying BPGM deficiency?

For genetic analyses of BPGM:

• Available testing methodologies:

  • Next-Generation Sequencing (NGS)/Massively parallel sequencing for comprehensive mutation detection

  • Deletion/duplication analysis to identify larger structural variants

  • Single gene sequencing for targeted analysis

• Sample requirements:

  • Multiple specimen sources have been validated:

    • Peripheral whole blood

    • Buccal swab

    • Saliva

    • Isolated DNA

    • Cell culture

    • Fresh or frozen tissue

    • Fetal samples (cord blood, fetal blood)

• Clinical context:

  • Testing is available for diagnosis of BPGM deficiency

  • Can be used for mutation confirmation in research settings

  • May require healthcare provider ordering in clinical contexts

• Research applications:

  • Correlating genetic variants with antibody-detected protein expression

  • Investigating transcriptional regulation through promoter region analysis

  • Studying hypoxia-responsive elements in the BPGM gene (~2000 bp upstream, 1000 bp downstream of the Transcription Start Site)