LARP6 Antibody, HRP conjugated

What is LARP6 and why is it important in research?

LARP6 (La ribonucleoprotein domain family member 6) is a critical RNA-binding protein that regulates the coordinated translation of type I collagen alpha-1 and alpha-2 mRNAs (CO1A1 and CO1A2). In humans, the canonical protein consists of 491 amino acid residues with a calculated molecular mass of 54.7 kDa, though it typically appears at approximately 70 kDa in Western blots due to post-translational modifications . LARP6 shuttles between the cytoplasm and nucleus, playing essential roles in translation and mRNA stability . Also known as Acheron or death-associated LA motif protein (ACHN), LARP6 belongs to the LARP superfamily of post-transcriptional regulators, all containing the characteristic La motif . Its importance in collagen biosynthesis makes it a significant target for research into fibrotic disorders, wound healing, and extracellular matrix regulation.

What are the main applications for LARP6 antibodies in research?

LARP6 antibodies are utilized across multiple experimental platforms with varying sensitivity requirements:

HRP-conjugated LARP6 antibodies are particularly valuable for direct detection in Western blot and ELISA applications, eliminating the need for secondary antibodies and reducing background signal when properly optimized .

How do HRP-conjugated LARP6 antibodies differ from unconjugated versions?

HRP (horseradish peroxidase) conjugation provides direct enzymatic detection capability to LARP6 antibodies. While unconjugated antibodies require a secondary antibody step for detection, HRP-conjugated versions offer:

• Streamlined experimental workflows with fewer incubation and washing steps

• Reduced background signal by eliminating cross-reactivity from secondary antibodies

• Enhanced sensitivity through direct enzymatic amplification of signal

• Improved quantitative accuracy in applications like ELISA and chemiluminescent Western blotting

What controls should be included when using LARP6 antibodies in Western blot applications?

Proper controls are essential for reliable LARP6 detection using HRP-conjugated antibodies:

Positive Controls:

• Jurkat cells and mouse brain tissue have been validated for LARP6 expression

• Recombinant LARP6 protein for band position verification

• Lysates from cells overexpressing tagged LARP6

Negative Controls:

• LARP6 knockout or knockdown samples

• Pre-absorption of antibody with immunizing peptide

• Secondary antibody-only control (for unconjugated primary antibodies)

• Substrate-only control (especially important for HRP-conjugated antibodies)

Loading Controls:

• Standard housekeeping proteins (β-actin, GAPDH)

• Total protein staining methods (Ponceau S, Coomassie)

The observed molecular weight of LARP6 is typically around 70 kDa, which differs from the calculated 55 kDa due to post-translational modifications including phosphorylation at multiple sites (Ser348, Ser396, Ser409, Ser421, Ser447, and Ser451) .

How should researchers optimize detection conditions for HRP-conjugated LARP6 antibodies?

Optimization is critical for achieving specific signal while minimizing background:

• Antibody concentration: Titrate the HRP-conjugated antibody (typically starting at 1:1000 and testing 2-3 dilutions in both directions) to determine optimal signal-to-noise ratio

• Blocking optimization:

  • Test multiple blocking agents (BSA, non-fat milk, commercial blockers)

  • Note that milk-based blockers may contain phosphatases that can interfere with phospho-specific detection

  • For HRP-conjugated antibodies, casein-based blockers often provide lower background

• Substrate selection:

  • Enhanced chemiluminescence (ECL) for standard detection

  • Super-signal ECL for low abundance targets

  • TMB or DAB for colorimetric detection

• Incubation conditions:

  • Test both room temperature (1-2 hours) and 4°C (overnight) incubations

  • Include 0.05-0.1% Tween-20 in wash and antibody diluent buffers

• Exposure time optimization:

  • For digital imaging systems, capture multiple exposure times

  • For film, perform a time series of exposures (10 seconds to 5 minutes)

What sample preparation methods are critical for detecting native LARP6?

LARP6 detection requires careful sample preparation to preserve protein integrity and epitope accessibility:

• Lysis buffer selection:

  • RIPA buffer: Good for nuclear and cytoplasmic fractions

  • NP-40/Triton buffer: Gentler extraction, better for preserving protein complexes

  • Include phosphatase inhibitors to preserve phosphorylation states at Ser348, Ser396, Ser409, Ser421, Ser447, and Ser451

• Subcellular fractionation:

  • Since LARP6 shuttles between nucleus and cytoplasm, separate fractionation may be necessary to analyze compartment-specific functions

  • Non-denaturing detergents are recommended for studies of LARP6 complexes with proteins like SEC61

• Sample denaturation:

  • Standard boiling in Laemmli buffer (95°C for 5 minutes) works for most applications

  • For membrane preparations, longer denaturation may be necessary

  • Avoid freeze-thaw cycles which can degrade phosphorylated epitopes

• Protein concentration determination:

  • BCA or Bradford assay recommended

  • Load 20-50 μg total protein per lane for cell lysates, 50-80 μg for tissue extracts

How can LARP6 antibodies be used to study its interaction with the 5' stem-loop of collagen mRNAs?

LARP6 acts as a key regulator of type I collagen expression by binding to the 5' stem-loop (5'SL) structure in collagen mRNAs. Research approaches include:

• RNA-protein complex detection:

  • RNA electrophoretic mobility shift assays (REMSA) can be performed using cell extracts containing LARP6 and radiolabeled 5'SL RNA

  • Anti-LARP6 antibodies can be used in supershift assays to confirm LARP6 presence in the complexes

  • Two distinct complexes (C1 and C2) form when LARP6 binds to 5'SL RNA

• Complex composition analysis:

  • Immunoprecipitation with LARP6 antibodies followed by RNA extraction and RT-PCR

  • Co-immunoprecipitation to identify additional proteins in the complex

  • Research has shown that SEC61 translocon associates with LARP6 and 5'SL RNA in both C1 and C2 complexes

• Functional domain mapping:

  • SIM mutant of LARP6 forms only complex C1 and loses the ability to interact with SEC61 translocon

  • Combined use of mutant constructs and antibody detection can map critical functional domains

To detect these interactions, researchers should consider crosslinking approaches before immunoprecipitation to stabilize transient RNA-protein complexes.

What approaches can resolve contradictory LARP6 antibody data in experimental systems?

When faced with conflicting LARP6 antibody results, consider these methodological approaches:

• Epitope mapping:

  • Different antibodies target different regions of LARP6

  • N-terminal vs. C-terminal vs. middle region antibodies may yield different results

  • Compare immunogen sequences: some antibodies target the sequence "PLFPNENLPS KMLLVYDLYL SPKLWALATP QKNGRVQEKV MEHLLKLFGT FGVISSVRIL KPGRELPPDI RRISSRYSQV GTQECAIVEF EEVEAAIKAH EFMITESQ"

• Isoform recognition:

  • Up to 2 different isoforms have been reported for LARP6

  • Verify which antibody epitopes are present in each isoform

  • Design experiments to specifically distinguish between isoforms

• Post-translational modifications:

  • LARP6 is a phosphoprotein with eight identified phosphorylation sites

  • Six sites reside in the C-terminal domain: Ser348, Ser396, Ser409, Ser421, Ser447, and Ser451

  • Phospho-specific antibodies versus total LARP6 antibodies may give different results

  • Treatment with phosphatases before Western blotting can help resolve discrepancies

• Validation with orthogonal methods:

  • Complement antibody data with mRNA detection, tagged protein expression

  • CRISPR/Cas9 knockout controls for definitive specificity testing

  • Compare multiple antibodies from different vendors or different clones

How can researchers optimize LARP6 antibody-based detection in different species models?

LARP6 is conserved across species, but antibody reactivity varies:

For cross-species application:

• Sequence alignment:

  • Check immunogen sequence alignment with target species

  • The higher the conservation in the epitope region, the greater likelihood of cross-reactivity

• Titration requirements:

  • Higher antibody concentrations may be required for less conserved species

  • Start with 2-3× the recommended dilution for human samples

• Validation approaches:

  • Positive control from target species alongside human control

  • Competition with immunizing peptide

  • Knockdown/knockout controls in the relevant species

How should researchers address high background issues with HRP-conjugated LARP6 antibodies?

High background is a common challenge with HRP-conjugated antibodies that can be addressed through systematic optimization:

• Blocking optimization:

  • Increase blocking agent concentration (3-5% BSA or milk)

  • Extended blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Commercial blocking solutions specifically designed for HRP conjugates

  • Include 0.1-0.3% Tween-20 in wash and antibody diluent buffers

• Antibody dilution:

  • Further dilute HRP-conjugated antibody (1:5000-1:10000)

  • Reduce incubation time or temperature

  • Prepare fresh dilution for each experiment

• Membrane handling:

  • Increase washing steps (5-6 washes, 10 minutes each)

  • Use fresh transfer buffer and PVDF instead of nitrocellulose for lower background

  • Pre-wash membrane in methanol followed by water before blocking

• Enzyme inhibition:

  • Add 0.05% sodium azide to block endogenous peroxidase activity

  • Include levamisole to block alkaline phosphatase activity

  • For tissue sections, hydrogen peroxide pre-treatment

What strategies can minimize epitope masking when detecting LARP6 in protein complexes?

LARP6 functions in multi-protein complexes (particularly with SEC61 and in 5'SL RNA binding), which can complicate antibody accessibility:

• Sample preparation modifications:

  • Gentler lysis conditions using non-denaturing detergents for studying complexes

  • Brief sonication or benzonase treatment to reduce nucleic acid interference

  • Consider crosslinking followed by IP for transient interactions

• Epitope retrieval techniques:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Enzymatic retrieval using proteinase K or trypsin for fixed tissues

  • Denaturation in 8M urea before dilution and immunoprecipitation

• Alternative detection approaches:

  • Use of multiple antibodies targeting different epitopes

  • Alternative LARP6 antibodies targeting N-terminal vs. C-terminal regions

  • Native PAGE versus SDS-PAGE for complex integrity preservation

How can researchers distinguish between specific and non-specific bands when using LARP6 antibodies?

Distinguishing true LARP6 signal from non-specific bands requires rigorous controls:

• Molecular weight verification:

  • Calculated molecular weight: 54.7 kDa

  • Observed molecular weight: ~70 kDa (due to post-translational modifications)

  • Phosphorylated forms may appear as multiple bands or at slightly higher molecular weights

• Validation approaches:

  • Peptide competition assay using the immunizing peptide

  • LARP6 knockdown/knockout samples as negative controls

  • Comparison of multiple antibodies targeting different epitopes

  • Immunoprecipitation followed by mass spectrometry

• Band pattern analysis:

  • Be alert for isoforms (up to 2 reported for LARP6)

  • Phosphorylated versus dephosphorylated forms

  • Proteolytic degradation products

  • For HRP-conjugated antibodies, enzyme degradation can generate unexpected bands

• Documentation of species differences:

  • Mouse and rat LARP6 have high sequence homology to human (97% and 94% respectively)

  • Different band patterns may be observed in different species due to species-specific post-translational modifications or isoforms

How can LARP6 antibodies be utilized in RNA-protein interaction studies?

Advanced research into LARP6's RNA-binding properties requires specialized approaches:

• RNA immunoprecipitation (RIP):

  • LARP6 antibodies can precipitate the protein along with bound RNAs

  • Subsequent RT-PCR or sequencing identifies target RNAs

  • Particularly useful for studying collagen mRNA regulation

  • The 5'SL structure of collagen mRNAs has been identified as a critical binding region

• Cross-linking immunoprecipitation (CLIP):

  • UV cross-linking stabilizes transient RNA-protein interactions

  • LARP6 antibodies precipitate cross-linked complexes

  • Sequencing identifies precise binding sites

  • Critical for mapping the 5 nucleotides within single-stranded regions of 5'SL that contribute to high-affinity binding

• Binding affinity measurements:

  • Surface plasmon resonance (SPR) using purified components

  • Microscale thermophoresis (MST) for measuring binding constants

  • Fluorescence anisotropy for real-time binding studies

• In vitro reconstitution systems:

  • Using purified components to map minimal interaction requirements

  • Combination with site-directed mutagenesis to identify critical residues

  • Modification with phosphatase treatment to understand regulation

What are the considerations for multiplexing LARP6 antibodies with other markers?

Multiplexing allows simultaneous detection of LARP6 with other proteins:

• Antibody selection criteria:

  • Host species diversity (avoid using multiple rabbit antibodies)

  • Direct conjugation to different fluorophores or enzymes

  • For HRP-conjugated antibodies, consider tyramide signal amplification for sequential detection

• Optimized protocols for co-detection:

  • Sequential immunostaining with complete antibody stripping between rounds

  • Careful blocking to prevent cross-reactivity

  • Spectral unmixing for fluorescence applications

• Recommended multiplexing pairs:

  • LARP6 with SEC61 to study their interaction at the ER membrane

  • LARP6 with collagen markers to correlate regulatory activity

  • LARP6 with subcellular markers (nuclear, cytoplasmic) to track shuttling

  • LARP6 with phosphorylation markers to study activation state

• Image analysis considerations:

  • Colocalization quantification methods

  • Signal intensity normalization

  • 3D reconstruction for spatial relationship analysis

How do phosphorylation states affect LARP6 detection and function?

LARP6 phosphorylation critically regulates its activity and detectability:

• Identified phosphorylation sites:

  • Eight phosphorylation sites total

  • Six sites in C-terminal domain: Ser348, Ser396, Ser409, Ser421, Ser447, and Ser451

  • Phosphorylation status affects protein migration on SDS-PAGE (explaining the difference between calculated 55 kDa and observed 70 kDa)

• Detection strategies:

  • Phospho-specific antibodies for individual sites

  • Lambda phosphatase treatment to confirm phosphorylation-dependent mobility shifts

  • Phos-tag gels for enhanced separation of phosphorylated forms

• Functional implications:

  • Phosphorylation likely regulates LARP6 binding to 5'SL of collagen mRNAs

  • May control interaction with SEC61 translocon

  • Could regulate nuclear-cytoplasmic shuttling

  • Different phosphorylation patterns may explain complex formation differences observed in gel shift assays

• Experimental considerations:

  • Preserve phosphorylation status with phosphatase inhibitors during extraction

  • Use kinase/phosphatase treatments to manipulate phosphorylation

  • Consider membrane preparation methods to retain phosphorylated forms