FBXL22 Antibody, HRP conjugated

What is FBXL22 and what functional roles does it play in cellular processes?

FBXL22 (F-Box and Leucine-Rich Repeat Protein 22) functions as a substrate-recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin ligase complex. Its primary role involves promoting ubiquitination of sarcomeric proteins, specifically alpha-actinin-2 (ACTN2) and filamin-C (FLNC) . These proteins are critical structural components of the muscle sarcomere, suggesting FBXL22 plays an important role in muscle protein turnover and homeostasis.

In functional studies, FBXL22 knockdown in zebrafish embryos resulted in severely reduced cardiac contractility accompanied by pericardial edema, indicating its essential role in cardiac function . The protein appears to have tissue-specific functions, as its expression is dynamically regulated during myoblast differentiation and in response to muscle denervation.

What are the known isoforms of FBXL22 and how do they differ functionally?

Multiple FBXL22 isoforms have been identified, with the full-length form (Fbxl22-236) and a shorter splice variant (Fbxl22-193) being the most studied. Research has demonstrated these isoforms exhibit different effects when overexpressed in skeletal muscles:

Both isoforms induce different levels of protein degradation markers (total ubiquitin, p62, LC3B II), cytoskeletal protein alterations (dystrophin, desmin, vimentin), and changes in α-actinin isoform levels, suggesting they may target overlapping but distinct substrate pools .

How is FBXL22 gene expression regulated?

FBXL22 expression is regulated through multiple mechanisms:

• Developmental regulation: Expression increases during C2C12 myoblast differentiation

• Stress response: Upregulation occurs early following denervation in muscle atrophy models

• Transcriptional control: The FBXL22 promoter contains conserved regulatory elements including:

  • Two E-box elements (located between −145 and −70 of the proximal promoter)

  • A putative AP-2 consensus sequence

  • A putative CCAAT box

  • A putative FoxO binding element

Mutation of either E-box element results in significantly lower reporter gene activity, suggesting regulation by myogenic regulatory factors (MRFs) like MyoD1 and myogenin, which are elevated during muscle differentiation and neurogenic atrophy .

What experimental applications are suitable for HRP-conjugated FBXL22 antibodies?

HRP-conjugated FBXL22 antibodies are versatile tools applicable to multiple experimental techniques:

*For immunofluorescence, HRP-conjugated antibodies require an additional tyramide signal amplification (TSA) step to convert the enzymatic activity to a fluorescent signal .

The advantages of HRP-conjugated antibodies include elimination of secondary antibody incubation steps, reduced background from secondary antibody cross-reactivity, and potential signal amplification through the enzymatic activity of HRP.

What controls should be included when validating HRP-conjugated FBXL22 antibodies?

A comprehensive validation strategy should include:

• Positive tissue controls: Human placenta, mouse brain, and skeletal/cardiac muscle tissues show reliable FBXL22 expression

• Negative controls:

  • Omission of primary antibody

  • Tissues/cells with FBXL22 knockdown (RNAi approach has achieved 60-70% knockdown in gastrocnemius muscles)

  • Pre-absorption with immunizing peptide

• Specificity controls:

  • Western blot verification of predicted 27 kDa band

  • Comparison of staining patterns with non-conjugated FBXL22 antibodies

• Enzymatic activity control:

  • Substrate-only reaction to verify HRP functionality

  • Hydrogen peroxide inactivation control

Multiple commercial FBXL22 antibodies are raised against specific protein regions (e.g., AA 100-C-terminus or AA 123-229), which should be considered when selecting controls for validation experiments .

How can researchers optimize Western blotting protocols for HRP-conjugated FBXL22 antibodies?

Optimized Western blotting with HRP-conjugated FBXL22 antibodies requires attention to several key parameters:

Sample preparation:

• Include proteasome inhibitors (e.g., MG-132) in lysis buffers to prevent degradation

• Load 12-20 μg protein per lane (higher loading may be needed for low-expression tissues)

Electrophoresis and transfer:

• Use 4-20% gradient gels for optimal resolution around 27 kDa (FBXL22's predicted size)

• PVDF membranes provide better protein retention for potentially low-abundance FBXL22

Blocking and antibody incubation:

• Block with 3-5% nonfat milk in TBST for 1 hour at room temperature

• Dilute antibody in blocking solution (typically 1:1000-1:5000)

• Incubate overnight at 4°C for maximum sensitivity

Detection optimization:

• Use enhanced chemiluminescent substrate specifically formulated for HRP

• For low signals, consider substrate with extended signal duration

• Expose membrane multiple times with increasing durations

Troubleshooting note: If background is high, increasing washing steps (5x 5 minutes with TBST) and reducing antibody concentration often improves signal-to-noise ratio .

How can FBXL22 antibodies be used to study protein-protein interactions within the SCF complex?

Investigating FBXL22's role in the SCF complex requires specialized approaches:

• Co-immunoprecipitation (Co-IP): FBXL22 antibodies can precipitate the entire SCF complex, allowing identification of associated proteins. In published protocols:

  • Cell lysates are incubated with anti-FBXL22 antibody

  • Complexes are captured with Protein G beads

  • Western blotting detects SCF components (SKP1, CUL1) and potential substrates

• Proximity Ligation Assay (PLA): Combines antibody specificity with signal amplification to visualize protein interactions in situ:

  • Primary antibodies against FBXL22 and potential interacting proteins

  • Secondary antibodies with attached DNA probes

  • If proteins are in proximity (<40 nm), probes allow rolling circle amplification

  • Resulting fluorescent signal indicates interaction

• FBXL22 substrate identification: HRP-conjugated antibodies in combination with ubiquitination assays enable investigation of novel substrates:

  • Cells co-expressing FBXL22, substrate candidate, and ubiquitin are treated with proteasome inhibitors

  • Lysates are immunoprecipitated with substrate-specific antibodies

  • Western blotting with anti-ubiquitin antibodies reveals FBXL22-dependent ubiquitination

This approach has successfully demonstrated FBXL22-mediated ubiquitination of ACTN2 and FLNC, showing characteristic smears of higher molecular weight ubiquitinated forms in the presence of FBXL22 .

What methodological considerations are important when studying FBXL22 in muscle tissue sections?

Muscle tissue presents specific challenges for FBXL22 immunodetection:

Fixation optimization:

• For immunohistochemistry: 4% paraformaldehyde fixation followed by paraffin embedding works well for FBXL22 detection in pancreatic cancer and muscle tissues

• For immunofluorescence: 2-4% paraformaldehyde for 15-20 minutes preserves antigenicity while maintaining tissue architecture

Antigen retrieval requirements:

• Heat-induced epitope retrieval with citrate buffer (pH 6.0) for 15-20 minutes improves detection

• For frozen sections, methanol fixation (10 minutes at -20°C) can enhance accessibility of some epitopes

Background reduction strategies:

• High muscle endogenous peroxidase activity requires thorough blocking (3% H₂O₂, 10-15 minutes)

• Autofluorescence can be mitigated with Sudan Black B treatment (0.1% in 70% ethanol)

Co-localization studies:

• When examining FBXL22 relationship with sarcomeric proteins, counterstaining with α-actinin or filamin-C can provide contextual information

• For HRP-conjugated antibodies, sequential TSA labeling with inactivation steps between targets prevents cross-reactivity

Successful immunohistochemical detection of FBXL22 has been achieved in human pancreatic cancer tissue using 1:100 dilution of FBXL22 antibody .

How can researchers use FBXL22 antibodies to study the differential expression in normal versus pathological conditions?

FBXL22 antibodies provide valuable tools for investigating expression changes across physiological and pathological states:

Quantitative Western blot analysis:

• Standardize protein loading (20-30 μg total protein)

• Use stain-free technology or housekeeping proteins as loading controls

• Analyze band intensity with appropriate software, normalizing to total protein

• Compare expression across conditions (e.g., innervated vs. denervated muscle)

Immunohistochemical quantification:

• Use consistent staining protocols across all samples

• Employ automated image analysis with defined parameters:

  • DAB intensity thresholds

  • Nuclear/cytoplasmic segmentation

  • Positive cell counting algorithms

Tissue microarrays (TMAs):

• Efficiently compare FBXL22 expression across multiple samples

• Reduces technical variation by processing all samples simultaneously

• Enables high-throughput screening across tissue types or disease states

Research application example: FBXL22 knockdown in denervated mouse gastrocnemius muscles showed partial protection against atrophy with larger mean fiber cross-sectional area compared to controls, suggesting therapeutic potential in denervation-induced muscle wasting .

Why might HRP-conjugated FBXL22 antibodies show inconsistent results in tissue staining?

Inconsistent tissue staining can result from several factors:

What strategies can address non-specific banding patterns in Western blots using FBXL22 antibodies?

Non-specific bands with FBXL22 antibodies can be addressed through systematic optimization:

• Sample preparation refinement:

  • Use fresh protease inhibitor cocktails in lysis buffers

  • Centrifuge lysates at high speed (14,000g for 15 minutes) to remove debris

  • Consider nuclear/cytoplasmic fractionation if bands correspond to nuclear proteins

• Blocking optimization:

  • Test alternative blocking agents (5% BSA or commercial blockers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1% Tween-20 to blocking solution

• Washing protocol enhancement:

  • Increase wash buffer stringency (0.1% to 0.3% Tween-20 in TBS)

  • Extend wash times to 5x 10 minutes

  • Use fresh wash buffer for each step

• Band identification strategies:

  • Molecular weight analysis: FBXL22 should appear at approximately 27 kDa

  • Compare with positive controls (mouse brain, human placenta lysates)

  • Verify with knockdown controls when possible

For HRP-conjugated antibodies specifically, direct binding to SDS-PAGE and titration experiments can help determine optimal antibody concentrations that maximize specific signals while minimizing background.

How can researchers distinguish between FBXL22 isoforms in experimental studies?

Differentiating between FBXL22 isoforms requires careful methodological approaches:

Western blot resolution strategies:

• Use longer SDS-PAGE gels (15-20 cm) with 10-12% acrylamide concentration

• Run gels at lower voltage (80-100V) for extended separation

• Consider specialized buffer systems optimized for mid-range protein separation

Isoform-specific detection approaches:

• Select antibodies targeting differential regions between isoforms

• For close molecular weight isoforms (Fbxl22-236 and Fbxl22-193), 2D gel electrophoresis may provide better resolution

• Complement protein detection with RT-PCR using isoform-specific primers

Experimental validation methods:

• Express recombinant isoforms as size standards

• Use tissues with known differential expression patterns

• Employ isoform-specific knockdown and monitor antibody reactivity changes

In published studies, overexpression of Fbxl22-236 in muscle cells led to significantly elevated levels of both the full-length Fbxl22 and potentially the endogenous protein, allowing researchers to distinguish between isoforms based on molecular weight differences and expression patterns .

How can FBXL22 antibodies contribute to understanding muscle atrophy mechanisms?

FBXL22 antibodies provide crucial tools for investigating atrophy pathways:

• Expression monitoring during atrophy progression:

  • Western blot quantification shows FBXL22 upregulation early in denervation-induced atrophy

  • Immunohistochemistry can localize expression changes to specific muscle regions

  • Time-course studies can correlate FBXL22 levels with atrophy markers

• Target protein degradation analysis:

  • Co-immunoprecipitation with FBXL22 antibodies can identify novel substrates

  • Ubiquitination assays using FBXL22 antibodies can quantify target protein modification

  • Proteasome inhibition experiments can confirm FBXL22's role in protein turnover

• Therapeutic intervention assessment:

  • RNAi knockdown of FBXL22 provided partial muscle sparing in denervated mouse medial gastrocnemius muscles

  • Mean fiber cross-sectional area was significantly larger in FBXL22 RNAi transfected muscles compared to controls

  • Quantitative immunohistochemistry can measure effects of potential therapeutics on FBXL22 expression

This research direction is particularly promising as FBXL22 knockdown resulted in partial preservation of muscle cross-sectional area size after denervation, suggesting therapeutic potential in neurogenic atrophy conditions .

What role might FBXL22 play in cardiac pathophysiology and how can antibodies help investigate this?

FBXL22 appears critical for cardiac function based on several lines of evidence:

• Developmental cardiac phenotypes:

  • Zebrafish embryos injected with FBXL22 morpholino developed severely reduced cardiac contractility (91.8 ± 1.3% of injected embryos)

  • Heart failure signs including pericardial edema were observed

  • Control morpholino-injected embryos maintained normal cardiac performance

• Sarcomeric protein regulation:

  • FBXL22 mediates ubiquitination of crucial cardiac structural proteins (ACTN2, FLNC)

  • Immunoblotting with FBXL22 antibodies demonstrates dose-dependent ubiquitination of these targets

  • In vivo ubiquitination assays show FBXL22-facilitated polyubiquitination of both ACTN2 and FLNC

• Potential pathophysiological research applications:

  • Immunohistochemical comparison of FBXL22 expression in normal versus failing hearts

  • Analysis of FBXL22 expression/activity in models of cardiac hypertrophy

  • Investigation of FBXL22 promoter regulation during cardiac stress conditions

FBXL22 antibodies enable these investigations through expression quantification, substrate identification, and localization studies in cardiac tissues.

What novel E3 ligase-substrate relationships might be discovered using FBXL22 antibodies?

HRP-conjugated and conventional FBXL22 antibodies facilitate discovery of novel E3 ligase-substrate relationships through several methodological approaches:

• Immunoprecipitation-mass spectrometry (IP-MS):

  • FBXL22 antibodies can pull down the entire SCF complex and associated proteins

  • MS analysis identifies potential novel substrates

  • Comparison of results with and without proteasome inhibition reveals stabilized substrates

• Proximity-dependent biotin identification (BioID):

  • Fusion of biotin ligase to FBXL22

  • Biotinylation of proximal proteins during interaction

  • Streptavidin pulldown followed by MS identifies interaction partners

  • FBXL22 antibodies verify expression of the fusion protein

• Global protein stability profiling:

  • Compare proteome changes in FBXL22 overexpression versus knockdown conditions

  • FBXL22 antibodies confirm manipulation efficiency

  • Identify proteins whose stability is inversely correlated with FBXL22 levels

Beyond the known ACTN2 and FLNC substrates, research suggests FBXL22 may interact with TGF-β signaling pathways in muscle tissues. This is supported by microarray data showing alterations in TGF-β signaling pathway components in denervated muscle tissues where FBXL22 expression is upregulated .