Leghemoglobin Lb120-8 Antibody

What is Leghemoglobin Lb120-8 and what is its function in plant biology?

Leghemoglobin Lb120-8 is an oxygen-binding plant hemoglobin found in the root nodules of Pisum sativum (garden pea). Its primary function is to provide oxygen to bacteroids within the root nodules while simultaneously protecting nitrogenase from oxygen-induced denaturation. This role is essential for symbiotic nitrogen fixation .

The protein is composed of a protoporphyrin IX (heme group) and a single peptide (globin). Its amino acid sequence varies depending on the legume species, while the heme group remains constant regardless of plant species or bacterial strain . Structurally, Leghemoglobin Lb120-8 has a predicted molecular weight of 19.9 kDa and is identified by the UniProt accession number Q9SAZ1 .

How are antibodies against Leghemoglobin Lb120-8 typically produced?

Antibodies against Leghemoglobin proteins are typically produced using recombinant proteins as immunogens. For example, Leghemoglobin A (LBA) antibodies are produced by immunizing rabbits with recombinant Leghemoglobin from sources such as Glycine max (soybean). The typical production process involves:

• Expression of recombinant Leghemoglobin in an appropriate system (E. coli, yeast, etc.)

• Purification of the recombinant protein

• Immunization of host animals (commonly rabbits)

• Collection and purification of antibodies using techniques such as Protein G chromatography

For Leghemoglobin Lb120-8 specifically, antibodies would be raised against the recombinant protein expressed in systems such as E. coli with appropriate tags (e.g., N-6xHis tag) .

What are the common applications for Leghemoglobin Lb120-8 antibodies in research?

Leghemoglobin antibodies are primarily used in the following research applications:

• Western Blotting (WB): For detecting and quantifying Leghemoglobin in protein extracts from root nodules or recombinant expression systems

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative analysis of Leghemoglobin levels

• Immunohistochemistry: For localization studies in plant tissues

• Flow Cytometry: For some applications involving cell-based studies

These antibodies enable researchers to study nitrogen fixation processes, plant-microbe interactions, and oxygen transport mechanisms in legume nodules. They are also valuable for validating recombinant Leghemoglobin expression in various systems being developed for biotechnological applications .

What are the optimal conditions for using Leghemoglobin Lb120-8 antibodies in Western blotting?

For optimal Western blotting with Leghemoglobin Lb120-8 antibodies, the following protocol is recommended:

Sample Preparation:

• Extract proteins using a modified post-alkaline method: Pellet cells from culture, wash with H₂O, resuspend in NaOH for 5 minutes at room temperature, pellet again, and resuspend in SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 0.1% Bromophenol blue, 10% Glycerol, 1% 2-Mercaptoethanol)

• Boil samples and centrifuge to collect the supernatant

Electrophoresis and Transfer:

• Load 10-20 μg of protein per lane on a 12-15% SDS-PAGE gel

• Note that Leghemoglobin may exhibit faster mobility than expected based on its calculated molecular weight (19.9 kDa), often appearing below 15 kDa

• Transfer to a PVDF or nitrocellulose membrane using standard protocols

Antibody Incubation:

• Block membrane with 3-5% non-fat dry milk or BSA in TBST

• Incubate with primary antibody at an optimized dilution (typically 1:1000 to 1:5000) in blocking buffer overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence reagents

Special Considerations:

• For recombinant His-tagged Leghemoglobin, anti-His antibodies may be used as an alternative detection method

• Optimal dilutions should be determined experimentally for each application and antibody lot

How can researchers distinguish between different leghemoglobin isoforms using antibodies?

Distinguishing between different leghemoglobin isoforms requires careful consideration of antibody specificity and complementary analytical methods:

Antibody Selection Strategies:

• Isoform-specific antibodies: Choose antibodies raised against unique peptide regions that differ between isoforms

• Epitope mapping: Determine the specific binding region of available antibodies to assess cross-reactivity potential

Complementary Analytical Methods:

• Isoelectric Focusing (IEF): Separate leghemoglobin isoforms based on their different pI values before immunodetection. The search results indicate that Lb isoforms can be separated by IEF, though some isoforms with similar pI values (differing by only 0.01 units) may be difficult to fully resolve

• Mass Spectrometry Validation: Use MALDI-TOF/MS to confirm the identity of specific isoforms after separation

  • "All bands containing Lbs were carefully excised from the IEF gels and the proteins were eluted and analyzed by MALDI-TOF/MS"

• 2D-Electrophoresis:

  • First dimension: IEF to separate by pI

  • Second dimension: SDS-PAGE to separate by molecular weight

  • Followed by Western blotting with specific antibodies

When using these approaches, researchers should be aware that different Lb isoforms (Lb a, Lb c, Lb d, etc.) have distinct characteristics, and their relative abundance may depend on the age of the nodules .

What considerations are important when designing immunological experiments to study leghemoglobin modifications?

When studying leghemoglobin modifications, several important considerations should guide experimental design:

Types of Modifications to Consider:

• Heme modifications: Leghemoglobin can form green derivatives with nitrated hemes under certain conditions

• Post-translational modifications: These may affect antibody recognition

• Redox state changes: Leghemoglobin can exist in different oxidation states (Lb II, Lb III)

Experimental Design Considerations:

• Sample Preparation:

  • Use protease inhibitors (e.g., PMSF) to prevent protein degradation

  • Consider anaerobic handling to preserve native redox states

  • Include appropriate controls for each modification state

• Detection Methods:

  • Spectroscopic analysis: Monitor characteristic absorption peaks (411 nm Soret region, 541 nm, 575 nm for oxygenated Lb)

  • EPR spectroscopy: For detecting species like dinitrosyl iron complexes (DNICs)

  • Immunoblotting: Using antibodies specific to modified or unmodified forms

• Validation Approaches:

  • Mass spectrometry to confirm specific modifications

  • Pyridinehemochromogen method for quantifying heme content

  • Use of oxidizing and reducing agents to generate reference standards

How can researchers optimize expression systems for producing recombinant Leghemoglobin Lb120-8 as antibody targets?

Optimizing expression systems for Leghemoglobin Lb120-8 production involves several strategies:

Host Selection:

• E. coli systems:

  • Advantages: Well-established, high yield potential, simple media requirements

  • Challenges: Ensuring proper folding and heme incorporation

  • Implementation: Use expression vectors with N-6xHis tag for purification

• Yeast systems (K. marxianus, P. pastoris):

  • Advantages: Better protein folding, higher yields, food-safe hosts (K. marxianus has GRAS status)

  • Implementation strategies from research:

    • Enhanced heme synthesis pathways through gene modification

    • Deletion of inhibitory genes (e.g., lsc1Δ ssn3Δ mutant)

    • Use of yeast artificial chromosomes (KmYACs) for large-scale heme synthesis modules

Expression Optimization:

• Heme biosynthesis enhancement:

  • "Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast"

  • Overexpress genes involved in heme biosynthetic pathway

  • Supply heme precursors in the medium

• Medium optimization:

  • Adjust glucose concentration based on dissolved oxygen levels (5-10%)

  • Optimize concentrations of glycine and FeSO₄·7H₂O

  • For E. coli: Consider supplementing with δ-aminolevulinic acid as a heme precursor

• Induction and harvest timing:

  • For K. marxianus: "The highest LBA production was achieved in 72 h of fermentation"

  • For E. coli: Optimal induction at mid-log phase with appropriate IPTG concentration

Using these approaches, researchers have achieved expression levels as high as 7.27 g/L of intracellular LBA in K. marxianus , making this an effective system for producing antibody targets.

What are the common challenges in developing and using Leghemoglobin Lb120-8 antibodies, and how can they be addressed?

Researchers face several challenges when working with Leghemoglobin Lb120-8 antibodies:

Challenge 1: Cross-reactivity with other hemoglobins

Solution approaches:

• Use peptide-specific antibodies targeting unique regions of Lb120-8

• Perform pre-absorption with related hemoglobins to increase specificity

• Validate antibody specificity using knockout/negative controls

• Consider using monoclonal antibodies for improved specificity

Challenge 2: Detecting low abundance leghemoglobins

Solution approaches:

• Implement signal amplification methods (e.g., biotin-streptavidin systems)

• Use more sensitive detection substrates for Western blotting

• Consider sample enrichment via immunoprecipitation before analysis

• Optimize antibody concentrations and incubation conditions

Challenge 3: Distinguishing between holo- and apo-forms

Solution approaches:

• Use spectrophotometric methods alongside immunodetection to confirm heme presence

• Develop antibodies specific to conformational epitopes present only in the holo-form

• Separate samples on native gels that preserve protein-heme interactions

• Use the pyridinehemochromogen method to quantify heme content in parallel

Challenge 4: Storage stability issues

Solution approaches:

• Store antibodies at -20°C with 50% glycerol to prevent freeze-thaw damage

• Aliquot antibodies to minimize freeze-thaw cycles

• For long-term storage, consider lyophilization approaches

• Monitor antibody functionality over time with positive controls

How can researchers verify the specificity of Leghemoglobin Lb120-8 antibodies in their experimental systems?

Verification of antibody specificity is crucial for reliable research outcomes. The following approaches are recommended:

Experimental Validation Methods:

• Positive and Negative Controls:

  • Positive: Recombinant Leghemoglobin Lb120-8 protein (commercially available)

  • Negative: Samples from non-legume plants or bacteroid-free nodule tissues

  • Competitive inhibition: Pre-incubation of antibody with purified antigen should abolish signal

• Cross-Reactivity Assessment:

  • Test against related leghemoglobin isoforms

  • Test against other hemoglobins (myoglobin, human hemoglobin)

  • Examine reactivity with samples from different legume species

• Molecular Weight Verification:

  • Confirm that detected bands match the expected molecular weight of Leghemoglobin Lb120-8 (approximately 19.9 kDa)

  • Note that some leghemoglobins may migrate faster than expected in SDS-PAGE

• Peptide Competition:

  • Pre-incubate antibody with the specific immunizing peptide

  • Signal elimination confirms specificity for the target epitope

• Orthogonal Detection Methods:

  • Confirm identity of immunodetected proteins by mass spectrometry

  • Use multiple antibodies targeting different epitopes of the same protein

What analytical methods can complement antibody-based detection of Leghemoglobin Lb120-8?

Several analytical methods can be used alongside antibody-based detection to provide complementary data:

Spectroscopic Methods:

• UV-Visible Spectroscopy:

  • Characteristic peaks for oxygenated Lb: 411 nm (Soret region), 541 nm, and 575 nm

  • Different forms of Lb have distinct spectral signatures

  • Can detect the formation of Lb-bound dinitrosyl iron complexes (DNICs)

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Useful for studying Lb-DNIC complexes with characteristic g-factors (g₁ = 2.04, g₂ = 2.03, g₃ = 2.014)

  • Can detect changes in Lb iron oxidation states

  • Low-temperature EPR spectroscopy allows detection of DNIC signals in both control and Lb-synthesizing bacteria

Mass Spectrometry Approaches:

• MALDI-TOF/MS:

  • For confirming protein identity and detecting modifications

  • "All bands containing Lbs were carefully excised from the IEF gels and the proteins were eluted and analyzed by MALDI-TOF/MS"

• Microelectrospray Ionization-Linear Ion Trap and Fourier Transform-Ion Cyclotron MS:

  • For detailed analysis of Lb heme modifications

  • Can analyze whole proteins when modified hemes are relatively unstable

Functional Assays:

• Peroxidase Activity Assays:

  • Lb displays peroxidase activity and catalyzes the reduction of organic peroxides

  • Can be measured using o-dianisidine as a reducing substrate

  • Provides functional verification alongside immunodetection

• Oxygen Binding Studies:

  • Measures the oxygen affinity of Lb proteins

  • Can distinguish between functional and non-functional Lb forms

How can Leghemoglobin Lb120-8 antibodies be used to study plant-microbe interactions in nitrogen fixation?

Leghemoglobin Lb120-8 antibodies offer powerful tools for investigating plant-microbe interactions in nitrogen fixation:

Spatial and Temporal Expression Analysis:

• Immunohistochemistry Applications:

  • Visualize Lb distribution within nodule zones (outer cortex, central zone, inner cortex)

  • Study the correlation between Lb expression and bacteroid distribution

  • Monitor changes in Lb localization during nodule development

• Expression Timing Studies:

  • Track Lb synthesis relative to nodulation stages

  • "Synthesis begins immediately after nodulation initiation and just before nitrogenase synthesis"

  • Correlate Lb expression with other symbiotic markers

Functional Analysis:

• Stress Response Investigations:

  • Study how oxidative and nitrosative stress affect Lb levels and modifications

  • "The interaction of the expressed Lb with oxidative and nitrosative stress inducers was studied by enzymatic methods and spectrophotometry"

  • Examine formation of modified forms such as nitrosylLb or DNICs under stress conditions

• Oxygen Regulation Studies:

  • Investigate the relationship between Lb levels and oxygen concentration in nodules

  • Study how Lb contributes to the "oxygen diffusion barrier" established by the endodermis

  • Examine Lb's role in maintaining appropriate oxygen levels for bacteroid respiration

• Symbiotic Efficiency Analysis:

  • Correlate Lb abundance with nitrogen fixation rates

  • Study how different Lb isoforms contribute to symbiotic efficiency

  • Investigate the relationship between Lb expression and nodule senescence

What approaches can be used to investigate the impact of leghemoglobin modifications on its function using antibody-based methods?

Investigating leghemoglobin modifications and their functional implications requires specialized approaches:

Detection of Specific Modifications:

• Modification-Specific Antibodies:

  • Develop antibodies that specifically recognize modified forms (e.g., nitrated hemes)

  • Use these alongside general Lb antibodies to determine the ratio of modified to unmodified forms

• Combined Spectroscopic and Immunological Approaches:

  • Correlate spectroscopic signatures of modifications with antibody-detected protein levels

  • "Hemes of Lb a, Lb c, and Lb d had a m/z 616, as expected for protoheme, whereas those from Lb am, Lb cm, and Lb dm had a m/z..."

Functional Correlation Studies:

• Activity-Modification Relationships:

  • Measure peroxidase activity in conjunction with immunodetection of specific Lb forms

  • "The peroxidase activity differed greatly depending on the cell type...In the case of the actively synthesized Lb culture, the peroxidase activity in all variants of the experiment was almost an order higher"

• Stress Response Analysis:

  • Investigate how oxidative and nitrosative stress affects both Lb modification state and function

  • "DNICs were destroyed in the presence of t-BOOH...Hb-DNICs also decay under the action of t-BOOH and H₂O₂"

• Structure-Function Studies:

  • Use antibodies to immunoprecipitate different modified forms

  • Perform functional assays on the isolated forms to correlate modifications with specific activities

How can researchers use antibodies against Leghemoglobin Lb120-8 to evaluate the quality of recombinant protein production systems?

Antibodies against Leghemoglobin Lb120-8 are valuable tools for evaluating recombinant protein production systems:

Quantitative Assessment:

• Expression Level Determination:

  • Western blot analysis to quantify protein yield in different expression systems

  • "After optimizing the medium recipe...an intracellular LBA titer of 7.27 g/L was achieved in the engineered strain in a 5 L fermentor"

  • Compare band intensities to standard curves using purified Lb or reference proteins like lactoglobulin

• Time-Course Studies:

  • Monitor expression levels at different time points during fermentation

  • "The highest LBA production was achieved in 72 h of fermentation"

  • Optimize harvest timing for maximum yield

Qualitative Assessment:

• Protein Integrity Analysis:

  • Detect potential degradation products using antibodies

  • Assess the impact of different expression conditions on protein stability

  • "Deletion of VPS10 and PEP4, which are involved in the degradation of misfolded proteins in the vacuole, has been employed to reduce hemoglobin degradation"

• Post-Translational Modification Evaluation:

  • Compare modifications between native and recombinant forms

  • Assess the impact of host cell machinery on protein processing

• Heme Incorporation Assessment:

  • Use antibodies that distinguish between holo- and apo-forms

  • Correlate antibody-detected protein levels with spectroscopic measurements of heme content

  • "The consumption of heme might alleviate the feedback inhibition on the enzyme(s) within the heme biosynthesis pathway, thereby promoting heme synthesis"

System Optimization:

• Host Strain Comparison:

  • Compare expression levels across different engineered strains

  • "Among the eight mutants, the lsc1Δ mutant demonstrated the greatest improvement in heme and leghemoglobin production"

• Media Formulation Studies:

  • Assess impact of different media components on expression levels

  • "After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO₄·7H₂O, a heme content of 66.32 mg/L and an intracellular LBA titer of 7.27 g/L were achieved"