AMT1-5 Antibody

What are AMT1-5 antibodies and what epitopes do they typically recognize?

AMT1-5 antibodies refer to antibodies targeting the AMT1 family of high-affinity ammonium transporters in plants (particularly Arabidopsis thaliana). Custom-made phosphorylation-sensitive antibodies can detect the conserved threonine in the C-terminal peptide sequence GMDMT(p)RHGGFA of AMT1 proteins . This threonine residue is highly conserved across AMT1;1 through AMT1;4, though AMT1;5 is an exception . Researchers should note that antibody specificity may vary depending on the exact epitope targeted, with some antibodies being able to detect all AMT1 family members while others are specific to individual transporters or their phosphorylation states.

What is the recommended protocol for using AMT1-5 antibodies in Western blotting?

Based on established protocols, membranes should be blocked using TBS-T containing 1% (w/v) casein hydrolysate for 1 hour, followed by overnight incubation with the primary antibody (typically IgG from rabbit at 1:1000 dilution) . After three washing steps with TBS-T, incubate the membrane for 1 hour with secondary antibody (such as polyclonal IgG from goat conjugated to horseradish peroxidase at 1:5000 dilution). For detection, treat the membrane with ECL SuperSignal West Dura solution and image using an appropriate system such as an Odyssey Fc imager . For loading control, membranes can be stained with 0.1% (w/v) Ponceau S in 5% (v/v) acetic acid for 2 minutes followed by washing with distilled water .

How can researchers verify the subcellular localization of AMT transporters using antibodies?

Membrane fractionation through two-phase partitioning can be used to separate plasma membrane and endosomal membrane proteins. The enrichment of plasma membrane proteins in the upper phase can be verified using antibodies against known markers such as plasma membrane ATPase AHA2, while enrichment of endosomal membrane proteins in the lower fraction can be confirmed using antibodies against markers like DET3 (a subunit of vacuolar ATPase) and vacuolar pyrophosphatase (VPPase) . AMT1;2, for example, has been shown to be enriched in the plasma membrane fraction in both root and shoot tissues using specific antibodies .

How can AMT1-5 antibodies be used to study the phosphorylation-dependent regulation of ammonium transport?

Phosphorylation-sensitive antibodies targeting the conserved C-terminal threonine residue can detect changes in AMT1 phosphorylation status under different conditions. For quantitative analysis, researchers can measure band intensity using ImageJ or similar software . In experimental designs:

• Compare phosphorylation states between wild-type and regulatory mutants (such as abi1 or cipk23)

• Monitor temporal changes in phosphorylation after ammonium shock

• Correlate phosphorylation levels with ammonium uptake rates

For example, research has shown that ammonium shock heavily phosphorylates AMT1s, with differences in phosphorylation levels observable between wild-type and abi1 mutant plants .

What considerations are important when using AMT1-5 antibodies to investigate protein-protein interactions?

When studying AMT1 interactions with regulatory proteins, antibodies can be used to validate results from other protein interaction techniques. Important considerations include:

• Complementary approaches: Combine antibody detection with split-ubiquitin yeast assays or Bimolecular Fluorescence Complementation (BiFC) experiments to cross-validate interactions

• Domain mapping: When studying interaction domains using deletion mutants, use antibodies to confirm expression and localization of truncated proteins

• Phosphorylation effects: Assess how phosphorylation status affects protein interactions by comparing wild-type and phosphorylation site mutants

Research has demonstrated that ABI1 interacts with AMT1;1 and AMT1;2 at the conserved C-termini, with C-terminal deletions in AMT1;2 (but not AMT1;1) disrupting this interaction .

How can researchers use AMT1-5 antibodies in conjunction with genetic approaches to study functional redundancy?

Multiple approaches can be combined for comprehensive analysis:

• Mutant verification: Use specific antibodies to confirm the absence of target proteins in insertion mutant lines. For example, in quadruple (qko) or triple insertion lines lacking combinations of AMT1;1, AMT1;2, AMT1;3, and AMT2;1

• Complementation studies: In complementation lines, antibodies can verify the expression of the reintroduced AMT protein

• Knockdown efficiency: In RNAi or amiRNA lines targeting AMT transporters, antibodies can quantify the degree of protein reduction

• Compensatory effects: Determine whether the loss of one AMT transporter affects the expression or phosphorylation of others

These approaches have been successfully used to demonstrate the additive contributions of AMT1;1 and AMT1;3 to high-affinity ammonium uptake in nitrogen-deficient Arabidopsis roots .

What methodological approaches can optimize AMT1-5 antibody detection across different experimental conditions?

To optimize antibody detection across various experimental conditions:

• Sample preparation optimization:

  • For membrane proteins, effective solubilization is critical; use appropriate detergents

  • Include phosphatase inhibitors when studying phosphorylation states

  • Consider plant growth conditions that maximize AMT expression (e.g., nitrogen starvation)

• Signal-to-noise optimization:

  • Titrate antibody concentrations to determine optimal dilution ratios

  • Extend washing steps if background is high

  • For low-abundance proteins, consider signal amplification methods

• Quantification approaches:

  • Use internal loading controls appropriate for your experimental conditions

  • Consider normalizing to total protein (Ponceau S) rather than single housekeeping proteins

  • For reproducible quantification, include standard curves with known amounts of purified protein

How can researchers interpret contradictory results when studying AMT1 phosphorylation with antibodies?

When facing contradictory results:

• Verify antibody specificity: Different antibodies may recognize different epitopes or may be affected differently by post-translational modifications

• Consider temporal dynamics: Phosphorylation states can change rapidly; for example, AMT1 phosphorylation patterns differ significantly between plants examined immediately after ammonium shock versus 2.5 hours later

• Account for growth conditions: Nitrogen status of plants dramatically affects AMT expression and phosphorylation

• Examine regulatory contexts: Different regulators may operate under specific conditions; for instance, ABI1 affects AMT1 phosphorylation , while CIPK23 inhibits ammonium transport

What controls should be included when working with AMT1-5 antibodies?

A comprehensive experimental design should include:

• Positive controls: Wild-type plant samples known to express the target AMT protein

• Negative controls:

  • Genetic knockout lines lacking the target protein (e.g., amt1;1-1 amt1;3-1 amt2;1-1 amt1;2-1 quadruple mutant)

  • Primary antibody omission control

• Phosphorylation controls:

  • Samples treated with phosphatase

  • Phosphomimetic mutants (e.g., T→D substitutions)

• Loading controls:

  • Total protein staining (Ponceau S)

  • Membrane fraction controls (e.g., AHA2 for plasma membrane)

How can researchers differentiate between specific AMT1 isoforms using antibodies?

Differentiating between AMT1 isoforms requires careful consideration:

• Epitope selection: Target non-conserved regions unique to each isoform

• Validation approaches:

  • Test antibodies on single knockout lines for each AMT

  • Perform peptide competition assays with isoform-specific peptides

  • Use recombinant proteins of each isoform as standards

• Cross-reactivity assessment:

  • Particularly important for AMT1;5, which lacks the conserved threonine present in other AMT1 proteins

What are the best practices for studying AMT1 transporters in different plant tissues?

Tissue-specific considerations include:

• Extraction protocols:

  • Root tissues may require more stringent extraction conditions

  • Ensure complete membrane protein solubilization

• Expression patterns:

  • AMT1;1, AMT1;2, AMT1;3, and AMT2;1 are all expressed in roots

  • Consider tissue-specific expression when interpreting results

• Developmental timing:

  • Expression and phosphorylation patterns may vary with plant age

  • Standardize sampling times and developmental stages

• Signal enhancement:

  • For tissues with low AMT expression, consider concentration steps

  • Optimize exposure times based on expected abundance

The successful application of these practices has enabled researchers to demonstrate that AMT1;2 is localized primarily to the plasma membrane in both root and shoot tissues .

How can AMT1-5 antibodies be applied to study the twin-histidine motif and its role in substrate selectivity?

The twin-histidine motif has been identified as a core structure responsible for substrate deprotonation and isotopic preferences in AMT pores . Researchers can use AMT1-5 antibodies to:

• Study protein expression levels when investigating mutants with alterations to histidine residues (H219, H386 in AMT1;2 or H188, H342 in AMT2)

• Confirm the presence of mutant proteins in functional studies examining transport activity

• Investigate potential conformational changes affected by mutations in the twin-histidine motif

• Correlate structural changes with functional outcomes in ammonium/methylammonium transport

Research has shown that mutations in these histidine residues can significantly affect transport activity and substrate selectivity .

How can AMT1-5 antibodies contribute to understanding the abscisic-acid-dependent regulation of ammonium transport?

ABI1 (a protein phosphatase 2C) has been identified as a regulator of AMT1 activity through interaction with the AMT1 C-termini . Researchers can use AMT1-5 antibodies to:

• Quantify AMT1 phosphorylation levels in wild-type versus abi1 mutant plants

• Track temporal changes in phosphorylation following ABA treatment

• Investigate whether other PP2C family members affect AMT1 phosphorylation

• Determine tissue-specific effects of ABA on AMT regulation

Research has demonstrated that the abi1 knockdown mutant shows reduced methylammonium susceptibility and altered AMT1 phosphorylation patterns, suggesting direct involvement in nitrogen uptake regulation .