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25% off all ELISA kits

Use code ELISA25-262 for 25% off all ELISAs through November.

S-Nitrosylated XIAP ELISA Kit

Measures s-nitrosylated XIAP (IAP-3 / BIRC4) and total XIAP in human cell and tissue lysates, enabling precise assessment of XIAP s-nitrosylation by ELISA.

This assay can be developed for other species (mouse, rat, etc.) upon request. Contact techsupport@raybiotech.com.

Product Description

Specifications

Size	1 Plate Kit, 2 Plate Kit, 5 Plate Kit
Estimated Lead Time	4-8 weeks
Species	Human
Compatible Sample Types	Tissue Lysates, Cell Lysates
Accession Number	P98170
Gene Id	331
Gene Symbols	XIAP, API3, BIRC4, IAP3
Protein Name / Synonyms	E3 ubiquitin-protein ligase XIAP; Baculoviral IAP repeat-containing protein 4; IAP-like protein (ILP, hILP); Inhibitor of apoptosis protein 3 (IAP-3, hIAP-3, hIAP3); RING-type E3 ubiquitin transferase XIAP; X-linked inhibitor of apoptosis protein (X-linked IAP)
Solid Support	96-well Microplate
Design Principle	Sandwich-based
Method Of Detection	Colorimetric
Research Area	Post-Translational Modifications, Nitrosylation, Epigenetics
Modification	S-Nitrosylation
Shipping Type	Blue ice
Storage	-20°C

Introduction

The RayBio^® S-Nitrosylated-XIAP ELISA Kit uses a modified "biotin-switch" method to allow for the direct visualization of human S-Nitrosylated XIAP by ELISA. In this S-Nitrosylation biotin switch assay, unmodified free cysteines are first blocked. S-Nitrosylated cysteines are then selectively reduced for specific labeling with biotin-maleimide reagents, which irreversibly bind to the cysteine thiol that was S-Nitrosylated. Biotinylation of the newly formed thiol groups can then be detected by ELISA. An anti-pan XIAP antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells and XIAP present in a sample is bound to the wells by the immobilized antibody. The wells are washed and HRP-conjugated streptavidin is used to detect S-Nitrosylated XIAP. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of S-Nitrosylated XIAP bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

Quantify quickly and easily
Screen numerous different lysates without performing a Western Blot analysis
Minimal hands-on time, convenient, and non-radioactive material

Kit Components

Component	Size / Qty
Microplate Coated With Anti-Pan-XIAP Antibody	1 plate
Positive Control	1 vial
HRP-Conjugated Streptavidin	200 μL
Wash Buffer	25 mL
Assay Diluent	15 mL
Lysis Buffer	5 mL
TMB One-Step Substrate Reagent	12 mL
Stop Solution	8 mL
S-Nitrosylation Buffer A	12 mL
S-Nitrosylation Buffer B	3 mL
S-Nitrosylation Buffer C	5 mL
S-Nitrosylation Buffer D	3 mL
S-Nitrosylation Buffer E	12 mL
S-Nitrosylation Blocking Reagent	2 vials
S-Nitrosylation Reduction Reagent I	2 vials
S-Nitrosylation Reduction Reagent II	2 vials
S-Nitrosylation Labeling Reagent	2 vials

Additional Materials Required

Acetone (pre-chilled (-20°C))
Microplate reader capable of measuring absorbance at 450 nm
Protease and Phosphatase inhibitors
Precision pipettes to deliver 2 μL to 1 mL volumes
Adjustable 1-25 mL pipettes for reagent preparation
100 mL and 1 liter graduated cylinders
Log-log graph paper or computer and software for ELISA data analysis
Absorbent paper
Tubes to prepare positive control or sample dilutions
Distilled or deionized water
Shaker

Assay Procedure Summary

Prepare all reagents, samples and Positive Control as instructed.
Block unmodified free cysteines using prepared S-Nitrosylation Blocking Buffer at 50°C with gentle rocking.
Precipitate proteins with ice-cold acetone.
Wash protein pellet twice with cold acetone/water mixture.
Resuspend protein pellets in Buffers and adding Reducing and Labeling Reagents.
Precipitate proteins with ice-cold acetone.
Resuspend protein pellets.
Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
Add 100 μL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

Storage/Stability

The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.

Customization

This assay can be developed for other species (mouse, rat, etc.) upon request. Contact techsupport@raybiotech.com for more information.

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Excel (Sandwich-ELISA-Analysis-Tool.xlsm)

Introduction

Product Features

Quantify quickly and easily
Screen numerous different lysates without performing a Western Blot analysis
Minimal hands-on time, convenient, and non-radioactive material

Kit Components

Component	Size / Qty
Microplate Coated With Anti-Pan-XIAP Antibody	1 plate
Positive Control	1 vial
HRP-Conjugated Streptavidin	200 μL
Wash Buffer	25 mL
Assay Diluent	15 mL
Lysis Buffer	5 mL
TMB One-Step Substrate Reagent	12 mL
Stop Solution	8 mL
S-Nitrosylation Buffer A	12 mL
S-Nitrosylation Buffer B	3 mL
S-Nitrosylation Buffer C	5 mL
S-Nitrosylation Buffer D	3 mL
S-Nitrosylation Buffer E	12 mL
S-Nitrosylation Blocking Reagent	2 vials
S-Nitrosylation Reduction Reagent I	2 vials
S-Nitrosylation Reduction Reagent II	2 vials
S-Nitrosylation Labeling Reagent	2 vials

Additional Materials Required

Acetone (pre-chilled (-20°C))
Microplate reader capable of measuring absorbance at 450 nm
Protease and Phosphatase inhibitors
Precision pipettes to deliver 2 μL to 1 mL volumes
Adjustable 1-25 mL pipettes for reagent preparation
100 mL and 1 liter graduated cylinders
Log-log graph paper or computer and software for ELISA data analysis
Absorbent paper
Tubes to prepare positive control or sample dilutions
Distilled or deionized water
Shaker

Assay Procedure Summary

Prepare all reagents, samples and Positive Control as instructed.
Block unmodified free cysteines using prepared S-Nitrosylation Blocking Buffer at 50°C with gentle rocking.
Precipitate proteins with ice-cold acetone.
Wash protein pellet twice with cold acetone/water mixture.
Resuspend protein pellets in Buffers and adding Reducing and Labeling Reagents.
Precipitate proteins with ice-cold acetone.
Resuspend protein pellets.
Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
Add 100 μL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

Storage/Stability

The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.

Customization

This assay can be developed for other species (mouse, rat, etc.) upon request. Contact techsupport@raybiotech.com for more information.

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25% off all ELISA kits

25% off all ELISA kits

S-Nitrosylated XIAP ELISA Kit