SLC22A5 Antibody, HRP conjugated

What is SLC22A5 and what is its biological function?

SLC22A5, also known as OCTN2 (Organic Cation Transporter Novel 2), is an integral plasma membrane protein that functions as both an organic cation transporter and a sodium-dependent high-affinity carnitine transporter . The human SLC22A5 protein consists of 557 amino acid residues with a calculated molecular mass of approximately 62.8 kDa, though it is often observed at around 70 kDa in Western blot applications due to post-translational modifications .

SLC22A5 plays several critical physiological roles:

• Facilitates the active cellular uptake of carnitine by transporting one sodium ion with one molecule of carnitine

• Transports organic cations such as tetraethylammonium (TEA) without sodium involvement

• Essential for fatty acid metabolism through its role in carnitine transport

• Involved in intestinal transport of bacterial quorum-sensing peptides

SLC22A5 is highly expressed in the kidney, skeletal muscle, heart, and placenta . Mutations in the SLC22A5 gene cause systemic primary carnitine deficiency (CDSP), an autosomal recessive disorder manifested by hypoketotic hypoglycemia, acute metabolic decompensation, and later by skeletal myopathy or cardiomyopathy .

Proper storage and handling of SLC22A5 Antibody, HRP conjugated is crucial for maintaining its activity and specificity:

Storage Conditions:

• Store at -20°C for long-term storage

• Some products may be stored at 2-8°C for up to 12 months from the date of receipt

• Avoid repeated freeze-thaw cycles as this can degrade the antibody

Handling Precautions:

• Protect from light, as HRP conjugates are light-sensitive

• Do not freeze HRP-conjugated antibodies after reconstitution

• When working with the antibody, keep it on ice or at 4°C

Buffer Information:
Some products are supplied in buffers containing:

• PBS with 0.02% sodium azide and 50% glycerol, pH 7.3-7.4

• Preservatives such as 0.03% Proclin 300

For reconstituted antibodies, aliquoting into single-use volumes before freezing is recommended to avoid multiple freeze-thaw cycles .

How do I select the appropriate dilution of SLC22A5 Antibody, HRP conjugated for my experiment?

Selecting the optimal dilution for SLC22A5 Antibody, HRP conjugated requires consideration of several factors:

Standard Recommended Dilutions:

• For ELISA: 1:500 - 1:2000

• For Western Blot: 1:500 - 1:2000

• For Immunofluorescence: 1:50 - 1:200

Optimization Strategy:

• Start with the manufacturer's recommended dilution range

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Include appropriate positive controls (tissues with known SLC22A5 expression such as kidney or heart)

• Include negative controls (tissues or cells with low SLC22A5 expression)

• Select the dilution that provides the best signal-to-noise ratio

Sample-Specific Considerations:

• Samples with high SLC22A5 expression (kidney, heart) may require higher dilutions to prevent signal saturation

• Samples with low expression may require lower dilutions (higher antibody concentration)

• Different sample types (cell lysates vs. tissue extracts) may require different optimal dilutions

Additional Factors:

• Detection method sensitivity (chemiluminescence vs. chromogenic)

• Incubation time and temperature

• Buffer composition

A titration experiment is recommended when using the antibody for the first time with a new sample type or detection system .

SLC22A5 polymorphisms have been associated with various diseases, including rheumatoid arthritis and carnitine deficiency. Using SLC22A5 Antibody, HRP conjugated to investigate these relationships requires specific methodological approaches:

Genotype-Expression Correlation Study Design:

• Sample Preparation:

  • Genotype samples for known SLC22A5 polymorphisms (e.g., -207C>G (rs2631367) or c.-149G>A variant )

  • Use SLC22A5 Antibody, HRP conjugated to measure protein expression levels via ELISA or Western blot

  • Correlate genotypes with expression levels

• Statistical Analysis Approach:

  • Compare protein expression levels between different genotype groups

  • In research on rheumatoid arthritis, the -207C>G polymorphism showed significant associations with disease manifestations

  • The table below shows the distribution of SLC22A5 genotypes found in rheumatoid arthritis patients:

Data from study of 404 RA patients and 560 controls . NS = not significant

• Functional Impact Assessment:

  • For the c.-149G>A variant, researchers created reporter constructs to test translation efficiency

  • Combined with carnitine transport measurements to assess functional consequences

  • SLC22A5 Antibody, HRP conjugated can verify protein expression changes resulting from such variants

• Disease Correlation Methodology:

  • Extra-articular manifestations were diagnosed in 16.7% of SLC22A5 GG homozygous patients vs. 7.2% of CC homozygous patients

  • The frequency of extra-articular manifestations was two-fold greater in homozygous GG patients compared to C allele carriers (OR = 2.06, 95% CI: 1.11–3.85, p = 0.022)

  • Similar methodologies can be applied to other diseases potentially associated with SLC22A5 polymorphisms

By applying these methodological approaches, researchers can effectively use SLC22A5 Antibody, HRP conjugated to investigate relationships between SLC22A5 polymorphisms and disease susceptibility, progression, and treatment response .

What are the most effective validation methods to ensure specificity of SLC22A5 Antibody, HRP conjugated?

Validating antibody specificity is crucial for obtaining reliable and reproducible results. For SLC22A5 Antibody, HRP conjugated, several methodological approaches can be employed:

Genetic Validation Approaches:

• Knockout/Knockdown Controls:

  • Compare antibody signal between wild-type cells and those with SLC22A5 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9)

  • A specific antibody should show significantly reduced or absent signal in knockout/knockdown samples

• Overexpression Controls:

  • Compare antibody signal between normal cells and those overexpressing SLC22A5

  • Include tagged versions (e.g., FLAG, His) that can be detected with alternative antibodies for confirmation

Biochemical Validation Approaches:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunogen peptide (e.g., amino acids 42-142 for antibodies targeting this region)

  • Apply to duplicate samples alongside non-blocked antibody

  • Specific binding should be blocked by the peptide, resulting in signal reduction

• Multiple Antibody Comparison:

  • Use multiple antibodies targeting different epitopes of SLC22A5

  • Concordant results increase confidence in specificity

  • Compare HRP-conjugated with unconjugated versions to ensure conjugation hasn't affected specificity

Application-Specific Validation Protocol:

For Western Blot validation, the following protocol based on published methods can be adapted :

• Prepare protein lysates (20 μg) from tissues/cells with known SLC22A5 expression

• Resolve by SDS-PAGE and transfer to PVDF membranes

• Block in 5% non-fat milk/PBS with 0.1% Tween 20 (PBS-T)

• Incubate with SLC22A5 Antibody, HRP conjugated at optimized dilution

• Wash thoroughly with PBS-T

• Develop using ECL substrate and detect signal

• Strip and reprobe with antibodies against housekeeping proteins (β-actin) for normalization

Positive Control Selection:

• Human samples: MCF7, OVCAR3, A-549, DU145, SW480

• Mouse samples: mouse intestine, mouse kidney

• Verify the expected molecular weight (~70 kDa observed vs. 63 kDa calculated)

These validation strategies ensure that SLC22A5 Antibody, HRP conjugated is specific and reliable for particular experimental contexts, essential for generating reproducible and meaningful research outcomes.

How can SLC22A5 Antibody, HRP conjugated be used in multiplex analysis with other carnitine pathway components?

Multiplex analysis involving SLC22A5 Antibody, HRP conjugated allows researchers to investigate the complex interplay between carnitine transport systems and related pathways. Here are methodological approaches for effective multiplex analyses:

Multiplexed Western Blot Strategy:

• Sequential Blotting Approach:

  • Detect SLC22A5 first using the HRP-conjugated antibody

  • Document results thoroughly

  • Strip the membrane (mild stripping buffer to preserve membrane integrity)

  • Reprobe with antibodies against related transporters or interacting proteins

  • This approach works well for proteins of different molecular weights

• Chromogenic Multiplex Detection:

  • Use SLC22A5 Antibody, HRP conjugated with one chromogenic substrate (e.g., DAB, brown)

  • Use other antibodies with different enzyme conjugates (e.g., alkaline phosphatase) and contrasting substrates

Recommended Protein Combinations for Comprehensive Carnitine Transport Analysis:

Combined Protein-Function Analysis Methodology:

A comprehensive approach for studying carnitine transport systems can incorporate:

• Expression Analysis:

  • Detect SLC22A5 protein levels using HRP-conjugated antibody via ELISA or Western blot

  • Quantify relative to appropriate controls

• Transport Activity Assessment:

  • Measure carnitine transport using radioactive or fluorescently labeled carnitine

  • Follow protocols similar to those described for [³H]-ergothioneine uptake :

    • Preincubate cells in HBSS buffer at 37°C

    • Incubate with uptake buffer containing labeled substrate

    • Stop uptake after 15 min by washing with ice-cold HBSS

    • Measure uptake using appropriate detection methods

• Inhibition Studies:

  • Test the effects of various compounds on transport activity

  • Correlate with SLC22A5 protein expression

  • Example: HEPES inhibition of SLC22A5, SLC22A4, and SLC22A15 uptake at various concentrations (5-25 mM)

By employing these multiplex strategies, researchers can gain comprehensive insights into carnitine transport systems and their interactions with related metabolic pathways, providing a more complete understanding of SLC22A5 function in normal physiology and disease states .