At1g14910 Antibody

What is At1g14910 and why is it significant for plant biology research?

At1g14910 (also known as PICALM1b) is an Arabidopsis thaliana gene encoding an ANTH domain-containing protein that functions as an adaptor protein for clathrin-mediated endocytosis (CME) . This protein plays a critical role in the recycling of secretory vesicle-associated longin-type R-SNARE VAMP72 group proteins . At1g14910 specifically interacts with the SNARE domain of VAMP72 and with clathrin at the plasma membrane .

The significance of At1g14910 lies in its fundamental role in membrane trafficking and protein recycling in plant cells. Understanding its function provides insights into:

• Vesicular trafficking mechanisms in plants

• Regulation of plasma membrane protein dynamics

• Plant cellular response to environmental stimuli

• Developmental processes dependent on membrane protein recycling

Antibodies against At1g14910 serve as essential tools for investigating these processes, allowing visualization of protein localization, quantification of expression levels, and analysis of protein-protein interactions.

What are the available options for At1g14910 antibodies and their specifications?

Based on current commercial offerings, At1g14910 antibodies are available with the following specifications:

This antibody recognizes the ENTH/ANTH/VHS superfamily protein encoded by the At1g14910 gene and is specifically designed for use with Arabidopsis thaliana samples . While detailed specifications about antibody type (monoclonal vs. polyclonal) are not explicitly mentioned in the search results, researchers should contact manufacturers for complete information about:

• Immunogen details

• Applications validated (Western blot, immunofluorescence, etc.)

• Recommended working dilutions

• Storage requirements

• Lot-specific performance data

For specialized research needs, custom antibody generation services may be considered when commercial options are insufficient.

How should researchers design membrane protein extraction protocols for optimal At1g14910 antibody detection?

Detecting membrane-associated proteins like At1g14910 requires specialized extraction techniques. Based on established protocols for similar membrane proteins:

• Microsomal fraction isolation:

  • Homogenize plant tissue in extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 10% glycerol, 1mM EDTA)

  • Include protease inhibitors (1mM PMSF, 1μg/ml leupeptin, 1μg/ml aprotinin)

  • Centrifuge at 10,000g for 10 minutes to remove debris

  • Ultracentrifuge supernatant at 100,000g for 1 hour to pellet microsomes

  • Resuspend microsomal pellet in buffer containing mild detergent

• Detergent selection is critical:

  • For Western blotting: 1% Triton X-100 or 0.5% NP-40

  • For maintaining protein-protein interactions: 0.5% digitonin or 0.1% DDM

  • For complete solubilization: 1% SDS (not compatible with native immunoprecipitation)

• Membrane protein enrichment strategy:
  "We enriched for plasma membrane-localized BIK1 by isolating microsomal protein fractions from Col-0/pBIK1:BIK1-HA... which express 100-fold higher levels of BIK1 and differentially accumulate BIK1 protein compared to wild-type."

• Proteasome inhibition for detecting unstable proteins:
  "To increase protein abundance and allow us to potentially capture immune-induced ubiquitination, proteasomal machinery was inhibited with 50 μM MG-132 an hour before treatment."

This extraction approach maximizes recovery of membrane-associated At1g14910 while preserving its native state for antibody detection.

What are the recommended immunoprecipitation protocols for studying At1g14910 interactions?

For investigating At1g14910 protein interactions, particularly with VAMP72 family proteins or clathrin, the following optimized immunoprecipitation protocol is recommended:

• Sample preparation:

  • Extract proteins from 5-10g Arabidopsis tissue using a gentle buffer (50mM HEPES pH 7.5, 150mM NaCl, 10% glycerol, 1mM EDTA, 0.5% Triton X-100)

  • Include phosphatase inhibitors (1mM NaF, 1mM Na₃VO₄) to preserve phosphorylation status

  • Clear lysate by centrifugation at 14,000g for 15 minutes at 4°C

• Antibody coupling:
  "The antibody was first crosslinked to the agarose beads by washing..."

  • Crosslink At1g14910 antibody to Protein A/G beads using dimethyl pimelimidate (DMP)

  • This prevents antibody co-elution with the target protein

• Immunoprecipitation:

  • Incubate cleared lysate with antibody-coupled beads overnight at 4°C with gentle rotation

  • Wash beads 4-5 times with washing buffer (extraction buffer with reduced detergent)

  • For interaction studies, use more stringent washes to reduce false positives

• Elution strategies:

  • For Western blot: Boil beads in SDS sample buffer

  • For mass spectrometry: Use acidic glycine buffer (100mM glycine, pH 2.5) or competitive elution with peptide

• Controls:

  • Input control (5% of starting material)

  • IgG control (non-specific antibody of same isotype)

  • Antibody-only control (no lysate)

For analyzing At1g14910 interactions with VAMP72, reference this approach from published literature: "Anti-GFP and anti-His-tag antibodies for the coimmunoprecipitation analysis of PICALM1a and VAMP721 interaction were purchased from MBL (598 and PM032, respectively)."

How can researchers optimize Western blot conditions for At1g14910 detection?

Optimizing Western blot conditions for detecting membrane proteins like At1g14910 requires specific technical considerations:

• Sample preparation:

  • Add 2X SDS sample buffer (100mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue, 200mM DTT)

  • Heat at 70°C for 10 minutes (avoid boiling membrane proteins)

  • Load 20-50μg total protein per lane

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Run at 100V until samples enter resolving gel, then 150V

• Transfer conditions:

  • Transfer to PVDF membrane (superior for hydrophobic proteins)

  • Use wet transfer with 25mM Tris, 192mM glycine, 10% methanol, pH 8.3

  • Transfer at 30V overnight at 4°C for maximum efficiency with membrane proteins

• Blocking optimization:

  • Block with 5% non-fat milk in TBS-T (TBS + 0.1% Tween-20) for 1 hour at room temperature

  • For phospho-specific detection, use 5% BSA instead of milk

• Antibody incubation:

  • Primary: Dilute At1g14910 antibody 1:1000 in blocking solution, incubate overnight at 4°C

  • Secondary: HRP-conjugated anti-species antibody at 1:5000 for 1 hour at room temperature

• Detection and analysis:

  • Develop using enhanced chemiluminescence (ECL)

  • For quantification, use digital imaging within the linear range

  • "Data should be expressed as mean/median ± standard deviation."

• Controls and troubleshooting:

  • Include positive control (if available)

  • Use membrane protein loading control (e.g., H⁺-ATPase)

  • If non-specific bands appear, optimize antibody dilution or consider longer washing steps

For membrane proteins, search result emphasizes: "While writing the 'Results' section, numerical expressions should be written in technically appropriate terms. The number of digits (1, 2 or 3 digits) to be written after a comma (in Turkish) or a point (in especially American English) should be determined."

What methods are recommended for studying At1g14910's role in clathrin-mediated endocytosis?

To investigate At1g14910's function in clathrin-mediated endocytosis (CME), integrate these advanced techniques:

• Colocalization analysis:

  • Perform double immunofluorescence with At1g14910 antibody and clathrin markers

  • Use high-resolution confocal microscopy with deconvolution

  • Quantify colocalization using Pearson's correlation coefficient

  • Example analysis approach: "PICALM1a was found to retain its function (as described below) and performed..."

• Temporal analysis of endocytic events:

  • Combine At1g14910 antibody staining with endocytic tracers (FM4-64)

  • Perform time-course experiments to capture different stages of endocytosis

  • Correlate At1g14910 localization with clathrin-coated pit formation and vesicle internalization

• Genetic manipulation experiments:

  • Use At1g14910 knockout/knockdown lines

  • Complement with fluorescently tagged At1g14910 constructs

  • Measure endocytosis rates of known cargo proteins in these genetic backgrounds

• Ubiquitination analysis:
  "Microsomal protein fractions were digested with trypsin, and anti-K-ε-GG agarose beads were used to enrich ubiquitinated peptides by affinity binding. Ubiquitinated lysines were identified based on a shift of ~114 Da."

  • Investigate whether At1g14910 is regulated by ubiquitination

  • Use anti-K-ε-GG antibody enrichment to identify specific ubiquitination sites

  • Correlate ubiquitination status with endocytic activity

• Electron microscopy:

  • Use immunogold labeling with At1g14910 antibodies

  • Examine distribution relative to clathrin-coated structures

  • Quantify gold particle distribution at plasma membrane vs. endocytic vesicles

• Proteomic analysis of At1g14910 complexes:

  • Immunoprecipitate At1g14910 from different cellular fractions

  • Use mass spectrometry to identify interacting partners

  • Compare interactome under different conditions (e.g., stress responses)

These approaches collectively provide mechanistic insight into At1g14910's precise role in clathrin-mediated endocytosis in plant cells.

How can researchers investigate At1g14910's interaction with R-SNARE proteins?

Investigating At1g14910's interactions with R-SNARE proteins requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP) analysis:

  • Perform reciprocal Co-IPs (IP: At1g14910, WB: VAMP72 and vice versa)

  • Include appropriate controls (IgG, input, knockout lines)

  • Similar to published approaches: "Anti-GFP and anti-His-tag antibodies for the coimmunoprecipitation analysis of PICALM1a and VAMP721 interaction"

• Domain mapping:

  • Generate truncation constructs of At1g14910

  • Test interaction with R-SNAREs via Co-IP

  • Identify minimal interacting domains

  • Compare to established domain structures: "Our crystal structure delineates the three-dimensional architecture of the most membrane-proximal Ig domains d6 and d7 of CD22 (CD22... d6–d7) and reveals that m971 binds at the membrane-most base of CD22 to mediate its antileukemic effects."

• Binding affinity measurements:

  • Express and purify recombinant proteins

  • Use Surface Plasmon Resonance (SPR) to determine binding kinetics

  • "The binding affinity of m971 Fab to CD22 (d1–d7) was calculated to be in the low nanomolar range (25 nM; Fig. 4)"

• Mutagenesis:

  • Identify conserved residues at the interaction interface

  • Generate point mutations and test effects on binding

  • Similar to approach in : "As expected from our structural studies, the S H53A mutation in HCDR2 decreased the binding affinity of m971 Fab to CD22 by approximately 8-fold (K D = 207 nM), primarily because of a faster off-rate"

• Functional assays:

  • Monitor R-SNARE trafficking in At1g14910 mutant lines

  • Assess At1g14910-dependent R-SNARE recycling rates

  • Correlate molecular interactions with functional phenotypes

• Structural analysis:

  • If possible, pursue structural determination of At1g14910-SNARE complexes

  • Use antibodies to validate structural findings in cellular context

Data from these approaches should be presented following scientific standards: "Tables should be comprehensible, and a reader should be able to express an opinion about the results just at looking at the tables without reading the main text."

How should researchers quantify At1g14910 expression levels across different experimental conditions?

Quantification of At1g14910 expression requires rigorous analytical approaches:

• Western blot quantification:

  • Use digital image capture within linear detection range

  • Perform densitometry using ImageJ or similar software

  • Normalize to appropriate loading controls (membrane protein)

  • Calculate relative expression (fold change)

  • Present data as: "Data should be expressed as mean/median ± standard deviation. Data as age, and scale scores should be indicated together with ranges of values."

• Statistical analysis:

  • Perform minimum 3 biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Report exact p-values: "While writing p values of statistically significant data, instead of p<0.05 the actual level of significance should be recorded. If p value is smaller than 0.001, then it can be written as p<0.01."

• Immunofluorescence quantification:

  • Use consistent acquisition parameters

  • Measure mean fluorescence intensity in defined regions

  • Quantify subcellular distribution patterns

  • Present representative images alongside quantification

• Quantitative standards and controls:

  • Include standard curve if absolute quantification is needed

  • Use recombinant protein standards when available

  • Include positive and negative controls in each experiment

• Presenting quantitative data:
  "Number of tables in the manuscript should not exceed the number recommended by the editorial board of the journal. Data in the main text, and tables should not be repeated many times."

  Example Table: At1g14910 Expression Under Different Treatments

  Treatment	Expression Level (Fold Change)	Subcellular Localization	Function
  Control	1.00 ± 0.15	Primarily plasma membrane	Baseline endocytosis
  Salt stress (150mM NaCl)	2.34 ± 0.42*	Enhanced endosomal localization	Increased endocytosis
  Osmotic stress (300mM mannitol)	1.87 ± 0.31*	Plasma membrane and cytoplasmic vesicles	Moderate endocytosis
  Cold stress (4°C, 3h)	0.76 ± 0.22	Primarily plasma membrane	Reduced endocytosis

  * p < 0.01 compared to control (n=4 biological replicates)

This quantitative approach enables robust comparison of At1g14910 expression and function across experimental conditions.

What controls are essential when conducting protein-protein interaction studies with At1g14910 antibodies?

Protein-protein interaction studies with At1g14910 antibodies require rigorous controls:

• Input controls:

  • Analyze 5-10% of pre-immunoprecipitation lysate

  • Verify presence of both bait (At1g14910) and potential interactors

  • Establish baseline abundance for quantitative comparisons

• Negative controls:

  • Non-specific IgG from same species as At1g14910 antibody

  • Knockout/knockdown lines for At1g14910

  • Unrelated protein immunoprecipitation (similar abundance)

  • "Include negative controls (secondary antibody only, pre-immune serum)"

• Positive controls:

  • Known interactors (e.g., clathrin heavy chain for At1g14910)

  • Similar to established interactions: "Anti-GFP and anti-His-tag antibodies for the coimmunoprecipitation analysis of PICALM1a and VAMP721 interaction were purchased from MBL"

• Reciprocal immunoprecipitation:

  • Immunoprecipitate with antibody against interactor

  • Detect At1g14910 in precipitate

  • Confirms interaction bidirectionally

• Competition controls:

  • Peptide competition to block specific antibody binding

  • Overexpression of potential interactor

  • Domain deletion mutants to map interaction interfaces

• Specificity controls:

  • Test closely related proteins as specificity controls

  • Test interaction under different buffer conditions

  • Verify specific interaction is maintained under stringent washing

• Quantification standards:

  • Include standard curve for semi-quantitative analysis

  • Report relative enrichment compared to IgG control

  • Include biological replicates for statistical analysis

Example data presentation:

This comprehensive control strategy ensures that reported protein-protein interactions are specific and biologically relevant.

How should researchers troubleshoot issues with At1g14910 antibody detection in complex samples?

When encountering detection problems with At1g14910 antibodies, implement this systematic troubleshooting approach:

• No signal or weak signal:

  • Increase protein loading (up to 50-75μg)

  • Reduce antibody dilution (1:500 instead of 1:1000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use signal enhancement systems (biotin-streptavidin amplification)

  • Enrich target protein: "We enriched for plasma membrane-localized BIK1 by isolating microsomal protein fractions"

• Multiple bands or high background:

  • Increase blocking time and concentration

  • Extend washing steps (5×10 minutes)

  • Try alternative blocking agents (BSA vs. milk)

  • Purify antibody by affinity chromatography

  • Use peptide competition to identify specific bands

  • Consider antibody cross-linking: "The antibody was first crosslinked to the agarose beads"

• Inconsistent results:

  • Standardize protein extraction method

  • Implement strict sample handling protocols

  • Use fresh antibody aliquots

  • Document lot-to-lot antibody variation

  • Include positive control in each experiment

• Sample-specific issues:

  • For membrane proteins, optimize detergent concentration

  • For low-abundance proteins, use enrichment strategies

  • For potentially ubiquitinated proteins: "Proteasomal machinery was inhibited with 50 μM MG-132 an hour before treatment"

• Advanced troubleshooting:

  • Test antibody on overexpression lines

  • Separate membrane fractions before analysis

  • Consider native vs. denaturing conditions

  • Use proximity ligation assay for in situ detection

  • Pre-absorb antibody against knockout tissue lysates

Systematic approach to At1g14910 antibody optimization:

"If detecting multiple bands, consider using peptide competition to identify specific bands. For membrane proteins that may form aggregates, look for higher molecular weight bands as well."

By systematically addressing detection issues, researchers can optimize At1g14910 antibody performance for their specific experimental conditions.

How can researchers use At1g14910 antibodies to investigate developmental regulation of endocytic pathways?

At1g14910 antibodies can provide insights into developmental regulation of endocytosis through these advanced approaches:

• Developmental expression profiling:

  • Analyze At1g14910 levels across developmental stages

  • Compare different tissues and cell types

  • Correlate with endocytic activity markers

  • Document changes in subcellular distribution

• Co-expression analysis:

  • Perform co-immunostaining with developmental markers

  • Compare expression patterns with other endocytic machinery

  • Similar to approaches with other developmental proteins: "ASK1/ask1 ask2 plants grown under the same conditions showed significant variation in the stages of embryo development within a single silique, as observed from 3 to 10 DAF"

• Tissue-specific analysis:

  • Use tissue sectioning and immunohistochemistry

  • Compare At1g14910 expression in different cell types

  • Correlate with tissue-specific endocytic requirements

  • "The body plan of major embryonic structures, including the shoot meristem, cotyledon, radicle (embryonic root), and hypocotyl, is established early in embryogenesis"

• Genetic interaction studies:

  • Analyze At1g14910 expression in developmental mutants

  • Examine phenotypes of At1g14910 mutants during development

  • Look for genetic interactions with developmental regulators

• Hormone response analysis:

  • Examine At1g14910 expression after hormone treatments

  • Correlate with hormone-induced changes in endocytosis

  • Track receptor internalization dynamics

• Stress-responsive regulation:

  • Analyze At1g14910 expression under various stresses

  • Correlate with adaptive endocytic responses

  • Quantify changes in membrane protein turnover

Example data presentation format:

This developmental profiling approach provides insights into how endocytic machinery is regulated during plant growth and development.

What methodologies allow researchers to study At1g14910 phosphorylation and its impact on function?

Investigating At1g14910 phosphorylation requires specialized approaches:

• Phosphorylation detection:

  • Immunoprecipitate At1g14910 and perform Western blot with phospho-specific antibodies

  • Use Phos-tag SDS-PAGE to separate phosphorylated forms

  • Perform mass spectrometry analysis of immunoprecipitated At1g14910

  • Similar to approaches analyzing phosphorylation: "As the phospho-status of BIK1 has been shown to affect its regulation by both RHA3A/B and PUB25/26"

• Phosphorylation site mapping:

  • Enrich phosphopeptides using TiO₂ or IMAC

  • Analyze by LC-MS/MS to identify specific phosphorylation sites

  • Validate identified sites through site-directed mutagenesis

  • "Multiple sequence alignments of peptides spanning -10 to +10 amino- and carboxyl-"

• Kinase identification:

  • Use kinase inhibitors to determine kinase family

  • Perform in vitro kinase assays with candidate kinases

  • Test direct interaction between At1g14910 and candidate kinases

  • Examine phosphorylation in kinase mutant backgrounds

• Functional analysis of phosphorylation:

  • Generate phospho-null (Ser/Thr→Ala) and phospho-mimetic (Ser/Thr→Asp/Glu) mutants

  • Assess effects on protein localization, stability, and interactions

  • Measure impact on endocytic rates and cargo selection

  • "Whether RHA3A/B and PUB25/26 compete for these sites or ubiquitinate distinct lysines remains to be tested experimentally"

• Stimulus-dependent phosphorylation:

  • Analyze phosphorylation changes after treatment with signaling molecules

  • Track temporal dynamics of phosphorylation/dephosphorylation

  • Correlate with changes in endocytic activity

Example phosphorylation site analysis table:

This comprehensive approach reveals how phosphorylation regulates At1g14910 function in endocytic processes.

How can researchers integrate At1g14910 antibody studies with advanced imaging techniques?

Combining At1g14910 antibody detection with advanced imaging techniques offers powerful insights:

• Super-resolution microscopy:

  • Use STORM or PALM imaging with fluorophore-conjugated secondary antibodies

  • Achieve 20-30nm resolution of endocytic structures

  • Quantify nanoscale organization of At1g14910 at plasma membrane

  • Co-visualize with clathrin and cargo proteins

• Live-cell and fixed-cell correlative imaging:

  • Track fluorescently-tagged markers in live cells

  • Fix at specific timepoints and immunostain for At1g14910

  • Correlate dynamic events with protein localization

  • Similar to methodological approaches in comparable studies: "We generated transgenic plants expressing GFP-tagged PICALM1a, which was found to retain its function"

• Electron microscopy techniques:

  • Perform immunogold labeling with At1g14910 antibodies

  • Use correlative light and electron microscopy (CLEM)

  • Quantify distribution at ultrastructural level

  • Map precise localization relative to clathrin-coated structures

• Fluorescence recovery after photobleaching (FRAP):

  • Use GFP-tagged At1g14910 for dynamics

  • Validate with antibody staining at fixed timepoints

  • Measure protein mobility and membrane association

• Förster resonance energy transfer (FRET):

  • Combine with acceptor photobleaching

  • Validate protein-protein interactions in situ

  • Measure interaction distances at nanometer scale

• Advanced image analysis:

  • Use machine learning for unbiased analysis of localization patterns

  • Perform object-based colocalization analysis

  • Track endocytic events with single-particle analysis

  • Quantify spatial statistics of protein distributions

Example imaging protocol optimization table:

"For immunofluorescence, try using Fab fragments instead of whole IgG molecules" to achieve better penetration and reduced background in super-resolution applications.

This integration of advanced imaging with antibody detection provides unprecedented insights into At1g14910's role in membrane trafficking events.