2. Thermo Fisher Scientific
  3. I-BLOCK™ PROTEIN-BASED BLOCKING REAGENT

Thermo Fisher Scientific I-BLOCK™ PROTEIN-BASED BLOCKING REAGENT User guide

  • Hello! I've reviewed the product details for the I-Block Blocking Reagent and associated products like ELISA-Light, Southern-Light Southern-Star, and Western-Light Western-Star. This document covers its applications, preparation, and storage, highlighting its use in various assays and detection systems. I understand that I-Block is a casein-based reagent designed to ensure high signal-to-noise ratio. Do you have any specific questions about these products or the information within the document?
  • What is the recommended working concentration for I-Block on neutral or positively charged nylon membranes?
    Can I-Block be used for phosphoprotein detection?
    How should I store I-Block powder?
    How long can I store I-Block solutions?
Applera, Applied Biosystems, AB (Design), CDP-Star, CSPD and Tropix are registered trademarks, and ELISA-Light, I-
Block, Southern-Light, Southern-Star, Western-Light and Western-Star are trademarks of Applera Corporation or its
subsidiaries in the US and/or certain other countries. All other trademarks are the sole property of their respective owners.
For Research Use Only. Not for use in diagnostic procedures. Information is subject to change without notice.
© Copyright 2007 Applied Biosystems. All rights reserved. Printed in USA.
AB Lit. Stock No. 120DA07-01
I-BLOCK™
PROTEIN-BASED BLOCKING REAGENT
Description
I-Block™ reagent is a highly purified casein-based blocking reagent. Each lot of I-Block™ reagent is
screened for low level alkaline phosphatase contamination and is selected to provide the highest signal-
to-noise performance in applications employing 1,2-dioxetane enzyme substrates such as CSPD® or
CDP-Star® substrates for alkaline phosphatase. I-Block™ reagent provides superior blocking
performance in both microplate-based solution assays and membrane-based immunodetection assays.
Applications
I-Block™ blocking reagent is recommended for use with all Tropix® chemiluminescent membrane-
based detection kits and immunoassay applications. The suggested working concentration is 0.2%
(weight/volume) for detection of biotin or fluorescein-labeled nucleic acid on neutral or positively
charged nylon membrane. For immunoassays and immunodetection on membranes (neutral nylon,
nitrocellulose, and PVDF), a concentration of 0.2% (weight/volume) is also recommended. For
immunodetection on positively charged nylon membrane, a concentration of 3% is recommended.
Note: I-Block is not recommended as a blocking reagent for use with phosphoprotein detection, when
using anti-phosphoepitope antibodies with broad reactivity, as casein contains multiple phosphorylated
sites.
Physical State
I-Block™ reagent is supplied as a dry powder with a protein concentration greater than 95%.
Storage
I-Block™ powder may be stored in a tightly closed container at room temperature. I-Block™
solutions may be stored for up to one week at 4ºC if sodium azide (to 0.02%) is added.
Catalog Number
T2015 30g
The following products include I-Block™ reagent:
ELISA-Light™ Immunoassay System
Southern-Light™ and Southern-Star™ Nucleic Acid Detection Systems
Western-Light™ and Western-Star™ Immunodetection Systems
Applied Biosystems Phone: (800) 542-2369 or (781) 271-0045
35 Wiggins Avenue FAX: (508) 383-7855
Bedford, MA 01730 http://appliedbiosystems.com
I-BLOCK™
PROTEIN-BASED BLOCKING REAGENT
A. Preparation for membrane-based applications:
0.2% I-Block™ reagent 0.2 g
1X PBS or 1X TBS 10 mL 10X PBS or 10X TBS
0.1% Tween®-20 100 μL Tween®-20
deionized H2O to 100 mL
Add 10X PBS or TBS to H2O. Add I-Block™ reagent, microwave for 40 sec, then stir, or heat
on a hot plate. DO NOT BOIL. Add Tween®-20 after solution has cooled. The solution will
remain slightly opaque, but particles should be dissolved. Cool to room temperature for use.
*For protein detection on positively charged nylon, use 3% I-Block™ reagent (3 g/100 mL) in
1X PBS with 0.1% Tween®-20.
B. Preparation for solution-based applications:
0.2% I-Block™ reagent 0.2 g
1X PBS or 1X TBS 10 mL 10XPBS or 10X TBS
0.05% Tween®-20 50 μL Tween®-20
deionized H2O to 100 mL
Add 10X PBS or TBS to H2O. Add I-Block™ reagent, microwave for 40 sec, then stir, or heat
on a hot plate. DO NOT BOIL. Add Tween®-20 after solution has cooled. The solution will
remain slightly opaque, but particles should be dissolved. Cool to room temperature for use.
C. Stock Buffer Preparation
10X Phosphate Buffered Saline (PBS)* 10X Tris Buffered Saline (TBS)*
0.58 M Na2HPO4 82.3 g 0.2 M Tris-base 24.23 g
0.17 M NaH2PO4-H2O 23.5 g 1.37 M NaCl 80 g
0.68 M NaCl 40 g
Adjust pH to 7.6 with conc. HCl
Add deionized H2O to 1000 mL Add deionized H2O to 1000 mL
*Alternative PBS and TBS recipes may also be used.
(click Support link)
/