At1g17350 Antibody

What is the At1g17350 gene and what protein does it encode?

The At1g17350 locus in Arabidopsis thaliana encodes a protein that functions within plant cellular processes. Similar to other plant proteins like NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1) and ATG6 (AUTOPHAGY-RELATED PROTEIN 6), the protein encoded by At1g17350 may be involved in plant immunity or developmental pathways . For proper characterization studies, researchers typically use antibodies raised against specific peptide sequences of the target protein, similar to how antibodies against AGO1 are developed using KLH-conjugated N-terminal peptides of the Arabidopsis thaliana AGO1 protein .

How should At1g17350 antibody be stored and reconstituted for optimal performance?

For optimal performance, store lyophilized antibody at -20°C until ready for use. When reconstituting, add approximately 50 μl of sterile water to the lyophilized powder and allow it to dissolve completely. After reconstitution, make small aliquots to avoid repeated freeze-thaw cycles that can degrade antibody quality. Always spin tubes briefly before opening to collect any material that might adhere to the cap or sides . For long-term storage of reconstituted antibody, keep aliquots at -20°C and avoid storing diluted antibody solutions for extended periods.

What are the recommended applications for At1g17350 antibody?

Based on similar plant protein antibodies, the At1g17350 antibody would likely be suitable for multiple experimental applications including:

• Western blot (WB) analysis for protein detection

• Immunofluorescence (IF) for protein localization studies

• Chromatin Immunoprecipitation (ChIP) for protein-DNA interaction studies

• Immunolocalization (IL) for tissue-specific expression analysis

• Small-RNA-IP-Seq for studying small RNA interactions

The selection of application should be determined by your specific research question, with appropriate optimization of antibody concentration for each technique.

How should Western blot protocols be optimized for At1g17350 antibody detection?

For optimal Western blot results with At1g17350 antibody, follow this research-validated protocol:

• Transfer proteins to a membrane (PVDF or nitrocellulose) following SDS-PAGE separation

• Block membranes for 1 hour with 5% low-fat milk powder in TBS-TT buffer (0.25% TWEEN20; 0.1% Triton-X)

• Probe with primary At1g17350 antibody at a 1:10,000 dilution in blocking solution for 1 hour

• Wash membranes thoroughly with TBS-TT buffer (3-5 washes, 5 minutes each)

• Incubate with HRP-conjugated secondary anti-rabbit antibody at 1:10,000 dilution for 1 hour

• Wash thoroughly and develop using chemiluminescence detection

For plant samples, grinding tissue in liquid nitrogen followed by extraction in a buffer containing protease inhibitors is recommended to preserve protein integrity and enhance detection sensitivity.

How can I confirm the specificity of At1g17350 antibody in my experimental system?

To confirm antibody specificity, implement these validation approaches:

• Knockout/knockdown controls: Compare protein detection between wild-type plants and those with reduced At1g17350 expression (using T-DNA insertion lines, CRISPR/Cas9 editing, or RNA silencing approaches similar to amiRNA techniques used for ATG6)

• Peptide competition assay: Pre-incubate the antibody with excess immunogenic peptide before application to your samples; specific signal should be significantly reduced

• Multiple antibody comparison: If available, test another antibody raised against a different epitope of the same protein

• Heterologous expression: Detect recombinant At1g17350 protein expressed in a system lacking endogenous expression

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein

What is the optimal sample preparation method for immunofluorescence using At1g17350 antibody?

For immunofluorescence studies in plant tissues:

• Fix fresh tissue samples in 4% paraformaldehyde for 1-2 hours at room temperature

• Wash thoroughly with PBS (3-5 times, 10 minutes each)

• Permeabilize cells with 0.1-0.5% Triton X-100 in PBS for 15-30 minutes

• Block with 5% BSA or normal serum in PBS for 1 hour

• Incubate with At1g17350 antibody at 1:100 to 1:500 dilution overnight at 4°C

• Wash thoroughly with PBS (3-5 times, 10 minutes each)

• Incubate with fluorophore-conjugated secondary antibody at manufacturer's recommended dilution (typically 1:200 to 1:1000) for 1-2 hours at room temperature

• Counterstain nuclei with DAPI if desired

• Mount slides with anti-fade mounting medium

This protocol can be adapted for studying protein localization similar to how ATG6 and NPR1 colocalization was studied in plant cells .

How can At1g17350 antibody be used for studying protein-protein interactions?

For investigating protein-protein interactions involving the At1g17350 protein:

• Co-immunoprecipitation (Co-IP):

  • Prepare plant tissue lysate in a mild lysis buffer containing protease inhibitors

  • Pre-clear lysate with Protein A/G beads

  • Incubate pre-cleared lysate with At1g17350 antibody overnight at 4°C

  • Add Protein A/G beads and incubate for 2-4 hours

  • Wash beads thoroughly and elute bound proteins

  • Analyze by SDS-PAGE followed by Western blotting for potential interacting partners

• Bimolecular Fluorescence Complementation (BiFC):

  • Clone At1g17350 and candidate interacting protein into BiFC vectors

  • Co-express in plant cells (via Agrobacterium-mediated transformation)

  • Validate protein expression using the At1g17350 antibody by Western blot

  • Visualize fluorescence to confirm interaction

• Proximity Ligation Assay (PLA):

  • Use At1g17350 antibody alongside antibodies against suspected interacting proteins

  • Follow PLA protocol to visualize proximal proteins within cells

These approaches can reveal functional relationships similar to those observed between ATG6 and NPR1, which were shown to interact directly and synergistically enhance plant immunity .

What are the considerations for using At1g17350 antibody in Chromatin Immunoprecipitation (ChIP) experiments?

For successful ChIP experiments with At1g17350 antibody:

• Crosslinking and Chromatin Preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10-15 minutes

  • Quench with 0.125 M glycine

  • Extract and sonicate chromatin to fragments of 200-500 bp

  • Confirm fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A/G beads

  • Incubate pre-cleared chromatin with At1g17350 antibody (5-10 μg per reaction) overnight at 4°C

  • Add Protein A/G beads and incubate for 2-4 hours

  • Wash thoroughly with increasingly stringent buffers

  • Reverse crosslinks and purify DNA

• Controls:

  • Include no-antibody control (mock IP)

  • Include IgG control

  • Use positive control regions (known targets)

  • Include input chromatin samples

• Analysis:

  • Perform qPCR for candidate target regions

  • Or proceed with ChIP-seq for genome-wide analysis

This approach would be particularly valuable if At1g17350 is involved in transcriptional regulation, similar to how NPR1 interacts with transcription factors in the nucleus to activate expression of downstream target genes .

How can At1g17350 antibody be used to study protein stability and degradation pathways?

To investigate protein stability and degradation mechanisms:

• Cycloheximide Chase Assay:

  • Treat plant samples with cycloheximide (100 μM) to inhibit protein synthesis

  • Collect samples at different time points (0, 1, 2, 4, 8 hours)

  • Analyze At1g17350 protein levels by Western blot

  • Calculate protein half-life based on degradation rates

• Proteasome Inhibitor Studies:

  • Treat samples with MG115 (100 μM) or MG132 to inhibit proteasomal degradation

  • Compare At1g17350 protein levels in treated versus untreated samples

  • Determine if protein is subject to proteasomal degradation

• Autophagy Inhibitor Studies:

  • Treat samples with Concanamycin A (5 μM) or Wortmannin (30 μM)

  • Compare protein levels before and after treatment

  • Determine if protein is subject to autophagy-dependent degradation, similar to tests conducted for NPR1-GFP stability

• Cell-free Degradation Assays:

  • Prepare plant cell extracts containing native At1g17350 protein

  • Incubate extracts with various inhibitors under controlled conditions

  • Monitor protein degradation over time by Western blot

This experimental design can reveal regulatory mechanisms controlling At1g17350 protein levels, similar to studies showing that ATG6 can maintain the stability of NPR1 through autophagy-independent mechanisms .

What are common troubleshooting strategies for weak or absent signal when using At1g17350 antibody?

When facing weak or absent signal in experiments:

For immunofluorescence applications:

• Ensure proper tissue permeabilization

• Try antigen retrieval methods if signal is weak

• Optimize antibody concentration specifically for immunofluorescence

• Reduce autofluorescence with treatments like sodium borohydride or TrueBlack

How can I quantify At1g17350 protein expression levels accurately across different experimental conditions?

For accurate protein quantification:

• Sample Preparation Standardization:

  • Harvest tissues at the same developmental stage and time of day

  • Process all samples simultaneously using identical protocols

  • Include multiple biological replicates (minimum 3)

• Western Blot Quantification:

  • Include a standard curve of recombinant protein if available

  • Load equal amounts of total protein (verify by Ponceau S staining)

  • Use a housekeeping protein control (like Actin, GAPDH, or Tubulin)

  • Ensure signal is within linear detection range of your imaging system

• Normalization Methods:

  • Calculate relative expression as: (At1g17350 signal / housekeeping protein signal)

  • Or use total protein normalization methods like Stain-Free technology

• Statistical Analysis:

  • Perform appropriate statistical tests (t-test, ANOVA) on replicate data

  • Report mean values with standard deviation or standard error

• Expression Level Comparison Table:

  Treatment Condition	Relative At1g17350 Expression (Mean ± SD)	Statistical Significance
  Control (untreated)	1.00 ± 0.12	Reference
  Treatment 1	[Value] ± [SD]	p < [value]
  Treatment 2	[Value] ± [SD]	p < [value]
  Treatment 3	[Value] ± [SD]	p < [value]

This quantification approach allows for reliable comparison of At1g17350 protein levels across different experimental treatments.

What controls should be included when using At1g17350 antibody for various experimental applications?

For rigorous experimental design, include these essential controls:

• Western Blot Controls:

  • Positive control (tissue/cell type known to express At1g17350)

  • Negative control (knockout/knockdown line if available)

  • Loading control (housekeeping protein or total protein stain)

  • Molecular weight marker to confirm expected protein size

• Immunofluorescence Controls:

  • Secondary antibody-only control (omit primary antibody)

  • Peptide competition control (pre-absorb antibody with immunizing peptide)

  • Negative control tissue (knockout/knockdown line if available)

  • Positive control (tissue known to express the protein)

• ChIP Controls:

  • Input DNA control (pre-immunoprecipitation chromatin)

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

  • No-antibody control

  • Positive control regions (known binding sites if available)

  • Negative control regions (non-target genomic regions)

• Co-immunoprecipitation Controls:

  • IgG control precipitation

  • Reverse co-IP (precipitate with antibody against interacting protein)

  • Input sample (pre-immunoprecipitation lysate)

  • Negative control (non-interacting protein)

These controls ensure experimental validity and help differentiate specific signals from background or artifacts.