SRGAP2B Antibody, HRP conjugated

What is SRGAP2B and how does it differ from other SRGAP2 paralogs?

SRGAP2B is a human-specific paralog that emerged from the partial duplication of the ancestral SRGAP2A gene. It contains only the first 9 exons (out of 22 in SRGAP2A), resulting in a truncated protein consisting primarily of the extended F-BAR domain (F-BARx). SRGAP2B lacks the last 49 C-terminal amino acids of SRGAP2A's F-BARx domain and contains 7 unique C-terminal amino acids. While SRGAP2B shares high sequence similarity with SRGAP2C (another human-specific paralog), they differ in several non-synonymous mutations - specifically, SRGAP2C has unique mutations affecting five arginine residues not present in SRGAP2B .

What are the recommended applications for SRGAP2B antibody, HRP conjugated?

The SRGAP2B antibody, HRP conjugated, is primarily optimized for ELISA applications. The polyclonal antibody is raised in rabbit against recombinant human SRGAP2B protein (amino acids 79-150) and demonstrates specificity for the human protein. For other applications like western blotting, immunoprecipitation, or immunohistochemistry, researchers should validate the antibody's performance or consider non-conjugated variants of SRGAP2 antibodies, which have demonstrated efficacy in these applications .

How can I confirm the specificity of SRGAP2B antibody to distinguish it from SRGAP2A and SRGAP2C?

Confirming specificity requires multiple validation approaches. First, perform western blots with recombinant SRGAP2A, SRGAP2B, and SRGAP2C proteins to assess cross-reactivity. SRGAP2B appears as a lower molecular weight band (approximately 50kDa) compared to SRGAP2A (approximately 120kDa) when using N-terminal-directed antibodies. To distinguish SRGAP2B from SRGAP2C, which have very similar molecular weights, consider using epitope-tagged recombinant proteins as controls or performing immunoprecipitation followed by mass spectrometry. Additionally, testing in cell lines with endogenous expression of these paralogs versus cells where individual paralogs have been knocked out can provide definitive evidence of specificity .

What is the optimal protein extraction protocol for detecting SRGAP2B in human brain samples?

For optimal SRGAP2B protein extraction from human brain samples, a specialized protocol addressing its unique characteristics is necessary. Begin with tissue homogenization in a buffer containing 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and complete protease inhibitor cocktail. Include phosphatase inhibitors (10mM NaF, 1mM Na₃VO₄) to preserve post-translational modifications. Since SRGAP2B forms hetero-dimeric complexes with SRGAP2A that can be targeted for proteasomal degradation, add a proteasome inhibitor (e.g., 10μM MG-132) during extraction. This prevents artificial degradation during sample processing. Perform extractions at 4°C and minimize freeze-thaw cycles, as research has shown these proteins are intrinsically unstable .

How should I design experiments to study the differential effects of SRGAP2B versus SRGAP2C on neuronal development?

To compare SRGAP2B and SRGAP2C effects on neuronal development, implement a multi-tiered experimental approach. Begin with in vitro studies using primary cortical neuron cultures transfected with either SRGAP2B or SRGAP2C expression constructs (including appropriate controls like SRGAP2A and empty vectors). Analyze dendritic spine density, morphology, and maturation at multiple time points (DIV7, 14, 21, and 28) to capture developmental dynamics. For in vivo studies, utilize in utero cortical electroporation to express fluorescently-tagged SRGAP2B or SRGAP2C in layer 2/3 pyramidal neurons. Include co-expression of a cell-filling fluorescent protein (e.g., Venus) to visualize dendrites and spines. Analyze animals at different developmental stages into adulthood, as research has shown SRGAP2C has unique long-lasting effects on synaptic density compared to SRGAP2B. For mechanistic insights, include parallel experiments with the F-BAR domain and F-BAR Δ49 constructs to isolate the contribution of the truncation versus arginine mutations .

What controls are essential when using the SRGAP2B antibody in ELISA to ensure valid results?

When using SRGAP2B antibody in ELISA, multiple controls are essential for result validation. Always include a titration curve of recombinant SRGAP2B protein as a positive control to establish assay sensitivity and linear range. For specificity controls, include wells with recombinant SRGAP2A and SRGAP2C proteins to assess cross-reactivity. Implement a negative control using an irrelevant protein of similar size and nature. For validating results in complex samples, prepare extracts from cells or tissues with confirmed SRGAP2B expression and compare with samples where SRGAP2B has been knocked down or knocked out. Include an isotype control antibody (rabbit IgG-HRP) to determine non-specific binding. Finally, implement technical replicates (minimum triplicate wells) and biological replicates (minimum three independent experiments) to ensure statistical robustness .

How can I simultaneously detect SRGAP2A and SRGAP2B interactions in human neuronal cells?

To detect SRGAP2A-SRGAP2B interactions in human neuronal cells, employ a combined co-immunoprecipitation and proximity ligation assay (PLA) approach. For co-IP, use antibodies targeting distinct epitopes – an anti-SRGAP2 C-terminal antibody (exclusive to SRGAP2A) for immunoprecipitation and the N-terminal SRGAP2B antibody for detection on western blots. This avoids cross-reactivity issues. For in situ visualization, implement PLA using primary antibodies from different species: rabbit anti-SRGAP2B and mouse anti-SRGAP2A (C-terminal). The proximity signal will only appear when the proteins are within 40nm of each other, indicating direct interaction. To validate interaction specificity, express mutated versions of either protein that disrupt binding domains. Additionally, perform FRET (Förster Resonance Energy Transfer) analysis using fluorescently tagged proteins to quantify interaction dynamics in live neurons .

What are the critical factors affecting the stability of the SRGAP2B protein during experimental procedures?

SRGAP2B protein stability is affected by multiple factors that require specific handling protocols. First, temperature significantly impacts stability – always maintain samples at 4°C during extraction and processing, and store at -80°C for long-term preservation. Avoid repeated freeze-thaw cycles, as research has shown that SRGAP2B is intrinsically unstable. Buffer composition is crucial – include 10% glycerol and 1mM DTT to maintain protein folding. pH fluctuations can dramatically reduce stability; maintain buffers at pH 7.2-7.4. SRGAP2B forms heterodimers with SRGAP2A that are targeted for proteasomal degradation, so proteasome inhibitors (10μM MG-132) should be included in extraction buffers. Protein concentration also affects stability, with dilute solutions showing accelerated degradation; maintain concentrations above 0.1mg/mL when possible. Finally, mechanical stress during homogenization can disrupt protein integrity, so use gentle methods like Dounce homogenization rather than sonication .

How should I optimize immunohistochemistry protocols when SRGAP2B antibody shows high background in brain tissue sections?

To optimize immunohistochemistry with SRGAP2B antibody when facing high background in brain tissue sections, implement a systematic optimization approach. First, modify fixation protocol – reduce paraformaldehyde concentration to 2% and fixation time to 12-18 hours at 4°C, as excessive fixation can create artifactual binding sites. For antigen retrieval, compare citrate buffer (pH 6.0) versus TE buffer (pH 9.0), with the latter showing superior results for SRGAP2 family antibodies. Implement a dual blocking strategy: first block with 10% normal serum from the species of the secondary antibody for 2 hours, then add 0.3% Triton X-100 and 3% BSA overnight at 4°C. Reduce primary antibody concentration to 1:500 and extend incubation to 48-72 hours at 4°C with gentle agitation. Include 0.1% Tween-20 in all washing steps (minimum 5 washes, 10 minutes each). Finally, implement an additional quenching step for endogenous peroxidase using 0.3% H₂O₂ in 100% methanol for 30 minutes before blocking to reduce non-specific signal from endogenous peroxidases in brain tissue .

How do the functional differences between SRGAP2B and SRGAP2C impact experimental design when studying human brain evolution?

The functional differences between SRGAP2B and SRGAP2C necessitate specific experimental designs when studying human brain evolution. SRGAP2C has been shown to have a stronger and more persistent effect on synaptic development compared to SRGAP2B, largely due to its five specific arginine mutations. When designing experiments, include time-course analyses extending into adulthood, as SRGAP2C's effects on synaptic density persist longer than SRGAP2B's. For molecular evolution studies, implement sequence analysis focusing on positive selection at the specific arginine residues that differentiate SRGAP2C from SRGAP2B. When performing functional studies, use both paralogs alongside chimeric constructs that interchange the mutated regions to isolate which specific mutations drive phenotypic differences. For population studies, examine copy number variations of both genes, as SRGAP2B shows significantly more variation than the more fixed SRGAP2C, suggesting different evolutionary pressures. Finally, when using cellular models, compare effects in both neuronal and microglial contexts, as recent evidence suggests these paralogs may have distinct effects on different neural cell types .

What methodological approaches are most effective for studying SRGAP2B's role in proteasome-dependent degradation of SRGAP2A?

To study SRGAP2B's role in proteasome-dependent degradation of SRGAP2A, implement a comprehensive methodology focused on protein turnover and interaction dynamics. First, establish a pulse-chase experimental system using metabolic labeling with ³⁵S-methionine in neuronal cells expressing both proteins to quantify SRGAP2A half-life with and without SRGAP2B co-expression. Combine this with cycloheximide chase assays in the presence or absence of proteasome inhibitors (MG-132, bortezomib) to confirm proteasome dependency. For mechanistic insights, perform co-immunoprecipitation studies to isolate SRGAP2A-SRGAP2B complexes, followed by ubiquitin western blotting to detect poly-ubiquitination. Implement proximity ligation assays to visualize the co-localization of these complexes with proteasome components in situ. To identify specific ubiquitination sites on SRGAP2A, perform mass spectrometry analysis of immunoprecipitated protein complexes. Finally, generate SRGAP2B mutants with altered dimerization capacity to determine which domains are critical for targeting SRGAP2A for degradation .

How should researchers interpret SRGAP2B antibody signals in the context of copy number variations in the human population?

Interpreting SRGAP2B antibody signals in the context of human copy number variations (CNVs) requires careful analytical consideration. SRGAP2B exhibits significantly more CNVs in the human population compared to SRGAP2C, which has been more fixed during evolution. When quantifying SRGAP2B protein levels via western blot or ELISA, normalize data to both housekeeping proteins and SRGAP2A levels within the same sample. For population studies, implement parallel genomic analysis (qPCR or digital droplet PCR) to determine SRGAP2B copy number in each sample and correlate with protein expression. When analyzing brain tissue, perform region-specific analyses, as expression patterns may vary across different brain areas. In cases of SRGAP2B duplication, assess whether protein levels scale linearly with copy number or if post-transcriptional regulation mitigates the effect of additional gene copies. For functional studies, categorize samples based on copy number status and separately analyze each group. Finally, when detecting SRGAP2B in heterozygous deletion carriers, be aware that antibody signal may come from the remaining allele, potentially masking copy number effects at the protein level .

What methods are most effective for studying the neotenic features induced by SRGAP2B/C in human brain development?

To study neotenic features induced by SRGAP2B/C in human brain development, integrate cutting-edge methodologies spanning multiple disciplines. Implement human cerebral organoid models derived from iPSCs with CRISPR-engineered SRGAP2B/C knockout, knockin, or dosage variations. Conduct time-course analyses using real-time imaging with genetically encoded indicators for synaptic activity (SynaptoZip or SynTagMA) to track synaptic development dynamically. Apply single-cell multi-omics (transcriptomics, proteomics) to identify cell-specific molecular signatures associated with SRGAP2B/C expression. For structural analyses, implement super-resolution microscopy techniques (STORM, PALM) with multiplexed antibody imaging to visualize subtle changes in synapse morphology across development. To understand functional implications, use multi-electrode arrays to measure network activity in organoids with modified SRGAP2B/C expression. When comparing human and non-human primate developmental trajectories, use species-specific induced neurons to isolate evolutionary differences. Finally, implement spatial transcriptomics in developing human and non-human primate brain tissues to map SRGAP2B/C expression domains and their relationship to areas undergoing neotenic development .

How can advanced imaging techniques be optimized for distinguishing between SRGAP2B and SRGAP2C protein localization?

Distinguishing SRGAP2B from SRGAP2C protein localization requires specialized advanced imaging approaches due to their high sequence similarity. Implement expansion microscopy combined with highly specific antibodies targeting the unique arginine residues that differentiate SRGAP2C from SRGAP2B. For in vivo studies, use CRISPRa/CRISPRi to modulate endogenous gene expression with simultaneous fluorescent tagging. When using overexpression systems, employ split fluorescent protein complementation assays where protein interactions complete the fluorophore, allowing visualization of specific heterodimer combinations (SRGAP2A-SRGAP2B versus SRGAP2A-SRGAP2C). For super-resolution approaches, implement DNA-PAINT with exchange-PAINT capability using oligonucleotide-conjugated antibodies specific to each paralog, allowing multiplexed imaging below the diffraction limit. To detect changes in protein conformation, use FRET-based sensors designed to recognize the specific structural differences between SRGAP2B and SRGAP2C. Finally, for absolute quantification in tissue sections, implement quantitative immunofluorescence with multi-spectral imaging and linear unmixing to separate closely overlapping signals .

What experimental approaches should be employed to investigate the differential effects of SRGAP2B versus SRGAP2C on microglial function?

Investigation of differential effects of SRGAP2B versus SRGAP2C on microglial function requires specialized experimental designs integrating cellular, molecular, and functional readouts. First, generate human iPSC-derived microglia expressing either SRGAP2B or SRGAP2C and perform comprehensive phenotypic characterization including morphological analysis (3D reconstruction of process complexity), motility assessment (time-lapse imaging of process dynamics), and phagocytic function (quantitative assays using pH-sensitive fluorescent particles). For in vivo studies, use tamoxifen-inducible Tmem119-CreERT2 mouse lines crossed with conditional SRGAP2B or SRGAP2C expression lines to achieve microglia-specific expression. Analyze morphological complexity through 3D reconstruction of Iba1+ microglia and implement two-photon imaging in awake behaving animals to assess real-time surveillance capabilities. For molecular insights, perform cell-type-specific RNA-seq focusing on cytoskeletal genes and inflammatory pathways, as research has shown that SRGAP2-deficient microglia exhibit altered expression of genes involved in actin dynamics. Finally, implement functional assays measuring synaptic pruning efficiency, response to injury, and interaction with neurons expressing human SRGAP2A to determine how these human-specific paralogs modify fundamental microglial functions .

How should researchers troubleshoot non-specific signals when using SRGAP2B antibody in western blotting?

When troubleshooting non-specific signals with SRGAP2B antibody in western blotting, implement a systematic approach addressing multiple technical variables. First, optimize protein extraction by using RIPA buffer supplemented with 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS to improve solubility of membrane-associated proteins. For electrophoresis, implement gradient gels (4-15%) which provide better resolution across the relevant molecular weight range (40-60kDa where SRGAP2B is expected). During transfer, use PVDF membranes (0.2μm pore size) instead of nitrocellulose, with a stepped transfer protocol (1 hour at 30V followed by 30 minutes at 100V) to improve transfer efficiency. For blocking, compare 5% non-fat milk versus 3% BSA in TBS-T, as research has shown different blocking agents can significantly affect background with SRGAP2 antibodies. Implement sequential antibody dilution tests (1:500, 1:1000, 1:2000, 1:5000) while extending incubation time (overnight at 4°C) to determine optimal signal-to-noise ratio. Include peptide competition assays using the immunizing peptide (amino acids 79-150) to identify which bands are specific. Finally, for very problematic samples, implement immunoprecipitation before western blotting to enrich for SRGAP2B protein .

What are the recommended strategies for quantifying SRGAP2B expression levels in human brain tissue samples?

Quantifying SRGAP2B expression in human brain tissue requires specialized approaches addressing the unique challenges of this protein. Implement a multi-method strategy starting with absolute quantification using a recombinant SRGAP2B protein standard curve (5-100ng range) alongside tissue lysates. For western blot quantification, use infrared fluorescent secondary antibodies and scan with systems like Odyssey, which provide wider linear dynamic range than chemiluminescence. When analyzing tissue lysates, prepare matched samples from multiple brain regions to account for regional expression differences. Normalize SRGAP2B signal to multiple housekeeping proteins (β-actin, GAPDH, and neuron-specific enolase) to account for cellular composition variations. For transcript analysis, design primers spanning unique junctions of SRGAP2B to distinguish from other paralogs, and validate specificity using synthetic templates. When possible, implement droplet digital PCR for absolute copy number determination without standard curves. For tissue-level protein quantification, use capillary western immunoassays (Wes, Jess systems) which provide better quantitative linearity and reproducibility than traditional western blots. Finally, consider single-cell approaches to measure expression variability across different neural cell populations .

How do post-translational modifications affect SRGAP2B antibody detection, and how can researchers account for these effects?

Post-translational modifications (PTMs) significantly affect SRGAP2B antibody detection through multiple mechanisms that require specific experimental controls. The SRGAP2B protein undergoes several PTMs including phosphorylation, ubiquitination, and potentially SUMOylation that can mask epitopes or alter electrophoretic mobility. To account for these effects, implement parallel western blots with phosphatase-treated samples (lambda phosphatase, 400U/mL, 30 minutes at 30°C) to determine if phosphorylation affects detection. For ubiquitination analysis, include proteasome inhibitors (MG-132, 10μM) in extraction buffers and perform immunoprecipitation under denaturing conditions to preserve these modifications. When analyzing band shifts, include high-percentage gels (12-15%) to resolve small mobility differences. For distinguishing between PTM-mediated and splice variant-mediated size differences, combine with RT-PCR analysis of transcript variants. If the antibody epitope (amino acids 79-150) contains predicted modification sites, generate synthetic peptides with the relevant modifications and perform competitive binding assays. For comprehensive PTM mapping, implement immunoprecipitation followed by mass spectrometry analysis focusing on the antibody epitope region. Finally, when comparing samples across experimental conditions, always process and analyze them simultaneously to minimize technical variation in PTM detection .

How can SRGAP2B antibodies be utilized in studying neurodevelopmental disorders associated with 1q21.1 deletions/duplications?

SRGAP2B antibodies offer valuable tools for investigating neurodevelopmental disorders associated with 1q21.1 chromosomal abnormalities through multiple applications. Implement immunohistochemical analysis of post-mortem brain tissue from 1q21.1 deletion/duplication carriers to assess SRGAP2B expression patterns in affected regions. For functional studies, generate iPSC-derived neurons from patient samples and quantify SRGAP2B protein levels using validated antibodies, correlating with dendritic spine density and morphology. Develop a multiplexed immunofluorescence panel combining SRGAP2B antibody with markers for excitatory/inhibitory synapses (PSD-95, Gephyrin) to assess synaptic effects. For mechanistic insights, implement proximity ligation assays to quantify SRGAP2B-SRGAP2A complexes in patient-derived neurons compared to controls. Use CRISPR-engineered isogenic iPSC lines with various SRGAP2B copy numbers to isolate dosage effects on neuronal development. In mouse models, implement stereotactic injection of viral vectors expressing human SRGAP2B in doses mimicking patient CNVs, followed by behavioral and electrophysiological phenotyping. Finally, for biomarker development, explore whether SRGAP2B or its degradation products can be detected in patient cerebrospinal fluid as potential diagnostic markers .

What experimental considerations are critical when studying the evolutionary implications of SRGAP2B/C expression in human brain development?

Studying evolutionary implications of SRGAP2B/C in human brain development requires specialized experimental approaches addressing their unique evolutionary context. First, implement comparative expression analysis across human, chimpanzee, and macaque brain development using species-specific antibodies or RNA probes that distinguish between paralogs. For functional comparisons, develop parallel systems using human and non-human primate iPSC-derived cerebral organoids with SRGAP2B/C either knocked out or expressed in controlled stoichiometric ratios. Implement time-course analyses extending beyond early development into periods mimicking adolescence, as human neoteny extends developmental windows compared to other primates. For mechanistic insights, perform comprehensive proteomic interaction studies to identify species-specific binding partners of SRGAP2A and how these are modified by SRGAP2B/C. Use CRISPR-generated humanized mouse models expressing SRGAP2B, SRGAP2C, or both to assess their cooperative effects on brain development and cognitive function. Implement single-cell multi-omics in human fetal brain samples to map the temporal-spatial expression patterns of all SRGAP2 paralogs. Finally, develop computational models integrating structural biology data to predict how the specific arginine mutations in SRGAP2C confer unique functions compared to SRGAP2B in modulating human-specific features of brain development .

How should researchers design experiments to understand the contribution of SRGAP2B to human-specific features of synaptic development?

To understand SRGAP2B's contribution to human-specific synaptic development, design experiments with precise molecular controls and evolutionary context. Implement a comprehensive approach beginning with human-mouse chimeric cultures where human iPSC-derived neurons expressing fluorescently-tagged SRGAP2B are co-cultured with mouse neurons to assess human-specific synaptogenic properties. Develop CRISPR knock-in models where individual domains of SRGAP2B are humanized in mouse neurons to identify which regions drive human-specific phenotypes. For temporal dynamics, implement optogenetic or chemogenetic induction systems to activate SRGAP2B expression at specific developmental timepoints, revealing critical periods for its influence on synaptic formation and maturation. Perform comparative electrophysiology in human organoids with SRGAP2B knockout versus wildtype to assess functional consequences on synaptic transmission and plasticity. Implement lattice light-sheet microscopy with engineered split-fluorescent protein reporters to visualize SRGAP2B-SRGAP2A interactions at developing synapses in real-time. For translational relevance, analyze human brain samples across developmental timepoints using spatial transcriptomics to map SRGAP2B expression to specific neurodevelopmental processes. Finally, develop computational models integrating structural, functional, and evolutionary data to predict how SRGAP2B influences human-specific features of cortical circuit development .

What are the key quality control parameters for validating a new lot of SRGAP2B antibody?

Validating a new lot of SRGAP2B antibody requires comprehensive quality control addressing multiple performance parameters. Begin with ELISA titration against the immunizing peptide (amino acids 79-150 of SRGAP2B) to establish binding affinity and compare EC50 values with the reference lot (variation should be <20%). Implement western blot analysis using both recombinant SRGAP2B protein and human brain lysates, comparing band intensity, molecular weight specificity, and background levels. Calculate the signal-to-noise ratio using densitometry and ensure it falls within 80-120% of the reference lot. For specificity assessment, perform parallel western blots with recombinant SRGAP2A, SRGAP2B, and SRGAP2C, quantifying relative cross-reactivity. Test antibody performance in immunoprecipitation using defined protein mixtures with known SRGAP2B concentrations and calculate recovery efficiency. Assess lot-to-lot consistency in immunohistochemistry using standardized human brain sections and implement automated image analysis to quantify staining intensity and distribution patterns. Finally, verify HRP conjugation efficiency through enzymatic activity assays using standard substrates (TMB, ABTS) and spectrophotometric analysis, ensuring consistent conjugation ratio between antibody and enzyme .

How does buffer composition affect SRGAP2B antibody performance in different experimental applications?

Buffer composition significantly impacts SRGAP2B antibody performance across different applications through multiple mechanisms that require specific optimization. For ELISA applications using HRP-conjugated SRGAP2B antibody, phosphate-based buffers (PBS) outperform Tris-based systems, but must be maintained at pH 7.2-7.4, as HRP activity is highly pH-dependent. Include 0.05% Tween-20 to reduce non-specific binding, but avoid higher concentrations that may disrupt antibody-epitope interactions. For blocking, 1% BSA in PBS provides superior signal-to-noise ratio compared to casein or non-fat milk, which can contain phosphatases affecting HRP activity. During antibody incubation, add 1mM EDTA to chelate metal ions that can decrease HRP activity and cause background. For storage buffers, include 50% glycerol with 1mM sodium azide for non-conjugated antibody, but avoid azide with HRP-conjugated versions as it inhibits peroxidase activity. When using the antibody for immunoprecipitation, add 0.1% digitonin to standard RIPA buffer to better preserve SRGAP2B-SRGAP2A complexes. Finally, for western blotting applications, transfer buffers containing 10% methanol improve binding to membranes, while including 0.1% SDS in TBST washing buffers reduces background without compromising specific signal .

How should storage and handling protocols be modified to maximize the shelf-life of SRGAP2B antibody, HRP conjugated?

Maximizing shelf-life of SRGAP2B antibody, HRP conjugated, requires specialized storage and handling protocols addressing both antibody and enzyme stability factors. Store the antibody at -20°C in single-use aliquots (10-20μL) to minimize freeze-thaw cycles, as research demonstrates HRP-conjugated antibodies lose approximately 15% activity per freeze-thaw cycle. For buffer composition, maintain 50% glycerol concentration which prevents freezing damage to protein structure, and include 1% BSA as a stabilizing protein carrier. Critically, avoid sodium azide in storage buffers as it irreversibly inhibits HRP activity. Maintain pH between 7.0-7.5, as HRP activity decreases by 30% outside this range. For long-term storage, add phenylmethylsulfonyl fluoride (PMSF, 1mM) to inhibit serine proteases that can degrade antibodies. When handling, minimize exposure to strong light, particularly UV, which can damage both antibody and HRP components. Once thawed for use, store diluted working solutions at 4°C and use within 7 days, as HRP activity in dilute solutions decreases approximately 5% per day. For projects requiring consistent sensitivity over time, consider preparing calibration standards to normalize results across different usage periods .