LACTB Antibody

What is LACTB and where is it localized in cells?

LACTB is a mammalian active-site serine protein that evolved from bacterial penicillin-binding proteins. Despite this evolutionary relationship, LACTB has acquired novel biochemical properties in eukaryotes. It is specifically localized in the mitochondrial intermembrane space, where it polymerizes into stable filaments that can extend more than a hundred nanometers in length .

The mitochondrial localization of LACTB can be verified through several complementary techniques:

• Immunofluorescence microscopy with mitochondrial co-markers

• Subcellular fractionation followed by Western blotting

• Protease protection assays with digitonin permeabilization

• Immuno-electron microscopy with nanogold-coupled antibodies

These approaches have confirmed that LACTB is a soluble protein in the mitochondrial intermembrane space rather than an integral membrane protein .

What are the recommended applications for LACTB antibodies?

Based on validated protocols, LACTB antibodies are primarily recommended for:

For Western blotting applications, LACTB appears as a single 55 kDa band in crude extracts that segregates with the mitochondrial fraction during subcellular fractionation . For immunofluorescence, co-staining with mitochondrial markers (such as mtRFP) is recommended to confirm the specificity of the antibody .

How can I validate the specificity of a LACTB antibody?

Validation of LACTB antibody specificity should include:

• Positive control: Use tissues/cells known to express LACTB (widely expressed across mammalian tissues)

• Negative control: Include LACTB knockdown or knockout samples

• Subcellular fractionation: Confirm enrichment in mitochondrial fractions

• Molecular weight verification: Ensure detection of the expected 54-55 kDa band

• Cross-reactivity testing: Examine species reactivity (human-specific antibodies are available)

For advanced validation, consider mitochondrial subfractionation to verify intermembrane space localization using markers like AIF and Opa-1 as comparisons .

What expression patterns of LACTB are observed across normal and cancer tissues?

LACTB protein expression follows a distinct pattern that makes it relevant for cancer research:

• Expressed in 100% of normal mammary glands analyzed

• Significantly downregulated in 34-42% of breast cancer tissues

• Reduced protein levels in approximately 83% (15 out of 18) of breast cancer cell lines tested

• Downregulation does not correlate with any particular tumor type, grade, or size

This expression pattern supports LACTB's proposed role as a tumor suppressor, with its restoration in cancer cells capable of inhibiting proliferation in several breast cancer cell lines (HMLER, MCF7-RAS, HCC1806) while minimally affecting non-tumorigenic cells (HME, MCF10A, BJ1) .

How can LACTB antibodies be used to study the protein's tumor suppressor function?

LACTB has been identified as a tumor suppressor in breast and ovarian cancers. Methodological approaches using LACTB antibodies include:

• Comparative expression analysis:

  • Western blot analysis of LACTB protein levels across tumor vs. normal tissue samples

  • Immunohistochemistry on tissue microarrays containing multiple patient samples

  • Correlation with clinical parameters and patient outcomes

• Mechanistic investigations:

  • Monitor changes in LACTB expression during induced cell differentiation

  • Assess LACTB levels before and after EMT induction

  • Track LACTB expression in response to antiproliferative signals

• Functional studies:

  • Use antibodies to validate LACTB re-expression in gain-of-function studies

  • Monitor LACTB levels following knockdown/knockout in loss-of-function experiments

  • Correlate LACTB levels with markers of cell proliferation (Ki-67, EdU incorporation)

The tumor suppressor function appears to be dependent on proper mitochondrial localization, as evidenced by studies showing that LACTB mutants lacking proper targeting do not display growth inhibitory effects on cancer cells .

What techniques can be used to investigate LACTB's polymeric structures?

LACTB forms unique polymeric structures in the mitochondrial intermembrane space. Investigations can include:

• Electron microscopy approaches:

  • Whole-mount immuno-electron microscopy using anti-LACTB antibodies with gold particles

  • Negative staining electron microscopy to visualize filament structure

  • Cryo-electron microscopy for higher resolution structural analysis

• Biochemical methods:

  • Blue native electrophoresis to separate intact LACTB polymers

  • 2D blue native SDS/PAGE for mass identification

  • CsCl gradient fractionation (1.25-1.28 g/cm³) to isolate LACTB polymers

  • Mass spectrometry for protein identification

These techniques have revealed that LACTB forms filaments composed of globular subunits that migrate over a broad molecular mass range from 600 kDa to several MDa in native gel electrophoresis .

How can LACTB antibodies help investigate mitochondrial lipid metabolism changes?

LACTB affects mitochondrial lipid metabolism, particularly through regulation of phosphatidylserine decarboxylase. Research approaches include:

• Correlation studies:

  • Western blot analysis of LACTB and phosphatidylserine decarboxylase levels

  • Immunofluorescence co-localization studies with lipid metabolism enzymes

• Functional assays:

  • Immunoprecipitation of LACTB to identify interacting partners in lipid metabolism

  • Analysis of mitochondrial phospholipid composition following LACTB manipulation

  • Correlation between LACTB levels and phosphatidylethanolamine synthesis

• Mechanism investigations:

  • Monitor changes in mitochondrial morphology using immunofluorescence

  • Assess impact on mitochondrial membrane organization

  • Evaluate consequences for mitochondrial micro-compartmentalization

The relationship between LACTB, lipid metabolism, and cell differentiation provides a novel mechanistic link between mitochondrial function and cancer development .

What experimental design considerations are important when studying LACTB in cancer systems?

When investigating LACTB in cancer research settings, consider:

• Expression system selection:

  • Inducible expression systems (like tetracycline-responsive) are preferred

  • Note that doxycycline can affect mitochondrial function, so appropriate controls must include doxycycline-treated control cells

• Cell line considerations:

  • Not all cancer cell lines respond similarly to LACTB manipulation

  • Some lines (like MDA-MB-231) show resistance to LACTB's growth inhibitory effects

  • Wild-type versus mutant LACTB should be compared in the same cellular background

• Readout parameters:

  • Combine proliferation assays (EdU incorporation) with apoptosis markers (annexin V)

  • Include differentiation markers specific to the cancer type

  • Monitor both short-term and long-term effects (cell cycle changes vs. differentiation)

These considerations are critical for accurate interpretation of LACTB's biological effects, as responses vary based on cellular context and genetic background.

What controls are essential when using LACTB antibodies in experimental procedures?

For rigorous scientific investigation using LACTB antibodies, incorporate these controls:

• Technical controls:

  • Primary antibody omission control

  • Isotype control (matching rabbit IgG)

  • Concentration-matched secondary antibody controls

• Biological controls:

  • LACTB knockdown/knockout samples

  • Overexpression of tagged LACTB with detection via tag antibody

  • Comparison across multiple cell lines with documented LACTB expression

• Signal validation:

  • Peptide competition assay to confirm signal specificity

  • Comparison of multiple antibodies targeting different LACTB epitopes

  • Correlation with mRNA expression where appropriate

These controls are particularly important given LACTB's post-transcriptional regulation and the observation that mRNA levels may not always correlate with protein expression .

How should LACTB antibodies be optimized for immunofluorescence microscopy?

For optimal immunofluorescence results with LACTB antibodies:

• Fixation method:

  • 4% paraformaldehyde is recommended for preserving mitochondrial structure

  • Avoid methanol fixation which can disrupt mitochondrial morphology

• Permeabilization:

  • Use mild detergents (0.1-0.2% Triton X-100) to access the intermembrane space

  • For super-resolution applications, consider digitonin permeabilization to selectively permeabilize the outer mitochondrial membrane

• Co-staining recommendations:

  • Include mitochondrial marker (TOM20, MitoTracker, mtRFP)

  • Consider co-staining with other intermembrane space proteins (AIF, Opa-1)

  • When studying cancer differentiation, include relevant differentiation markers

• Signal amplification:

  • For low expression levels, consider tyramide signal amplification

  • For multi-color imaging, use spectral unmixing to resolve overlapping signals

The recommended antibody dilution range of 1:100-1:400 should be optimized for each specific application and cell type .

What factors influence successful western blotting with LACTB antibodies?

For optimal western blot detection of LACTB:

• Sample preparation:

  • Use mitochondrial enrichment for enhanced sensitivity

  • Include protease inhibitors to prevent degradation

  • For native studies, extract with non-denaturing conditions

• Gel selection:

  • Standard 10-12% SDS-PAGE for denatured samples

  • Native PAGE or BN-PAGE for polymer analysis

  • 2D BN-SDS/PAGE for complex resolution

• Transfer considerations:

  • PVDF membranes are preferred for higher protein binding capacity

  • Semi-dry transfer systems work well for the 54 kDa LACTB protein

  • For polymer studies, extend transfer time and reduce methanol concentration

• Detection optimization:

  • 1:1000 antibody dilution is recommended as starting point

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence provide sufficient sensitivity

  • For quantitative analysis, consider fluorescent secondary antibodies

These optimizations are crucial for accurate detection of LACTB's 54-55 kDa band and distinguishing it from non-specific signals.

How can LACTB antibodies contribute to cancer biomarker research?

LACTB's differential expression in cancer versus normal tissues suggests potential biomarker applications:

• Diagnostic approaches:

  • Immunohistochemistry panels including LACTB for tissue classification

  • Analysis of LACTB expression patterns across cancer progression stages

  • Correlation with established diagnostic markers

• Prognostic investigations:

  • Longitudinal studies correlating LACTB levels with patient outcomes

  • Multi-marker panels combining LACTB with other mitochondrial proteins

  • Stratification of patients based on LACTB expression patterns

• Predictive biomarker potential:

  • Analysis of LACTB expression as a predictor of treatment response

  • Investigation of LACTB restoration as a therapeutic strategy

  • Monitoring LACTB levels during treatment

The finding that LACTB expression is significantly downregulated in 34-42% of breast cancer tissues warrants further investigation of its biomarker potential .

What experimental approaches can link LACTB's evolutionary history to its current functions?

LACTB's evolution from bacterial penicillin-binding proteins raises interesting questions:

• Structural biology approaches:

  • Use antibodies to purify LACTB for crystallization studies

  • Compare homology models with bacterial homologs

  • Investigate the predicted coiled-coil segment that enables polymerization

• Functional conservation analysis:

  • Compare enzymatic activities with bacterial counterparts

  • Investigate the functional significance of the PBP fold

  • Examine conservation of the active site serine

• Evolutionary investigations:

  • Compare LACTB across species using cross-reactive antibodies

  • Analyze functional divergence across evolutionary lineages

  • Investigate selective pressures on LACTB-encoding genes

Homology modeling using the crystal structure of Streptomyces R61 D-alanyl-D-alanine carboxypeptidase has revealed that LACTB exhibits a characteristic PBP fold with an α/β region and an all-helical region, providing insights into its evolutionary relationships .