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Secondary Antibodies  >  Chemiluminescent Western Blot Kits
 

Fluorescent TrueBlot®: Anti-Rabbit IgG IRDye800®

Mouse Monoclonal eB182
Code

18-3216-32
Size

100 µL
Price

$179.00
Availability

Ships next business day.
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Rabbit TrueBlot® IP / Western Blot:

Jurkat cell lysate (0.5 ml of 1x10e7 cells/ml) was incubated with rabbit anti-human Stat1 and immunoprecipitated using Protein G, Protein A and Anti-Rabbit Ig IP Beads. Precipitate from 5x10e5 cells was subjected to electrophoresis, transferred to a PVDF membrane, and Western blotted with anti-Stat1 using Rabbit TrueBlot®: Anti-Rabbit IgG HRP
Western Blot of Fluorescent TrueBlot®: Anti-Rabbit IgG IRDye800.
Lane 1:  Rabbit IgG, Non-reduced.  
Lane 2:  Rabbit IgG, Reduced. 
Load:  50 ng per lane.
Primary antibody:  none.
Secondary antibody: Fluorescent TrueBlot®: Anti-Rabbit IgG IRDye800 at 1:1,000 for 60 min at RT.
Block:  MB-070 for 30 min at RT.
Predicted/Observed size:  160 kDa for Rabbit IgG, Non-reduced. Migrates at slightly higher molecular weight.
Other band(s): none.
Shipping & Handling Fees Apply.

Shipping:
$40.00 to United States
Handling:
$20.00
Synonyms: IR800, TrueBlot, IR800 TrueBlot ULTRA, IRDye800® TrueBlot, TrueBlot for IP/WB, TrueBlot for immunoprecipitation, TrueBlot for western blotting, Fluorescent TrueBlot, Rb TrueBlot

Fluorescent TrueBlot®: Anti-Rabbit IgG IRDye800® Properties

Description
Fluorescent TrueBlot®: Anti-Rabbit IgG IRDye800® - 18-3216-32
Host
Mouse
Target Species
Rabbit
Known Cross Reactivity
Rabbit
Clonality
Monoclonal eB182
Application
FLISA : 1:5,000 - 1:10,000
IF Microscopy : >1:5,000
Western Blot : 1:10,000 - 1:25,000
ImmunoPrecipitation: User Optimized
Other Dilution: FLUORESCENT POLARIZATION: 2 - 20 nMFRET: 2 - 20 nM
Product Type
Component
Kit Type
Immunoprecipitation Kit
Physical State
Lyophilized
Shipping condition
Ambient
Format
IgG
Label
FP Value
3.0 moles IRDye® 800 per mole of IgG
Concentration
1.0 mg/mL by UV absorbance at 280 nm
Buffer
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Reconstitution Volume
100 µL
Reconstitution Buffer
Restore with deionized water (or equivalent)
Preservative
0.01% (w/v) Sodium Azide
Stabilizer
10 mg/ml Polyethylene Glycol (PEG-8000)
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Lot#  

Fluorescent TrueBlot®: Anti-Rabbit IgG IRDye800® Description

Background
Rabbit IgG TrueBlot® is a unique IRDye800® conjugated anti-rabbit IgG immunoblotting (second step) reagent. Rabbit IgG TrueBlot® enables detection of immunoblotted target protein bands, without hindrance by interfering immunoprecipitating immunoglobulin heavy and light chains. It is easy to generate publication-quality IP/Fluorescent Western Blot data with Rabbit IgG TrueBlot®, simply substitute the conventional IR800 anti-rabbit IgG blotting reagent with Fluorescent Rabbit TrueBlot® Antibody IRDye800® and follow the prescribed protocol for sample preparation and immunoblotting. Rabbit IgG TrueBlot® is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot preferentially detects the non-reduced form of rabbit IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Rabbit IgG TrueBlot® with the 55 kDa heavy chains and the 23 kDa light chains of the immunoprecipitating antibody is minimized thereby eliminating interference by the heavy and light chains of the immunoprecipitating antibody in IP/Western blotting applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions. Fluorescent Rabbit TrueBlot® Antibody IRDye800® may also be used for detection in immunoassays that do not employ immunoprecipitation.
Storage Condition
Store vial at 4° C prior to restoration. For extended storage aliquot contents and freeze at -20° C or below. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
Application Note
Fluorescent Rabbit TrueBlot® Antibody IRDye800® is provided as a lyophilized powder. In order to conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mls). If used conservatively at 2.5mls/blot, cat. 18-3216-32 (100µL) will yield enough reagent for 200 blots. Note that there are three key procedural considerations: 1. Protein A or G should not be used for the immunoprecipitation. For immunoprecipitation, anti-rat IgG beads, or anti-rabbit IgG beads should be used for rat or rabbit immunoprecipitating antibodies, respectively. 2. Immunoprecipitate should be completely reduced. 3. Bovine Serum Albumin, at low concentrations, should be used as the blocking protein for the immunoblot. DO NOT USE MILK. Fluorescence technology is widely used to detect proteins. However, many common visible fluorophores often result in considerable background fluorescence in the visible range. Visible fluorophores are rarely used for membrane-based protein detection because of this high background. IRDye® 800 and IRDye® 700DX antibody and reagent conjugates are specifically designed for protein detection methods that use longer-wavelength, near-infrared (IR) fluorophores to visualize proteins in western blotting and other applications. Very low background fluorescence in the IR range provides for a much higher signal-to-noise ratio than visible fluorophores. Detection levels in the picogram range on Western blots rival the sensitivity of chemiluminescence on film. IRDye® 800 conjugates are optimized for the Odyssey® Infrared Imaging System developed by LI-COR. IRDye® 800 conjugates are also suitable for immunofluorescence microscopy using commercially available excitation/emission filters in the 780nm/820nm range. Dual simultaneous labeling in western blots or microscopy is achieved when IRDye® 800 conjugates are used in conjunction with IRDye® 700DX or DyLight™680 conjugates. IRDye® 800 and IRDye® 700DX conjugates provide an ultra-sensitive and convenient alternative to standard chemiluminescent protein detection methods, as well as a valuable tool for multicolor imaging.
Purity/Specifity
Fluorescent Rabbit TrueBlot® Antibody IRDye800® Conjugate was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by Immunoelectrophoresis resulted in a single precipitin arc against anti-Rabbit Serum. Reactivity is observed against native Rabbit IgG by both Western blot and ELISA.
Disclaimer Note-General
This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information. All products of animal origin manufactured by Rockland Immunochemicals are derived from starting materials of North American origin. Collection was performed in United States Department of Agriculture (USDA) inspected facilities and all materials have been inspected and certified to be free of disease and suitable for exportation. All properties listed are typical characteristics and are not specifications. All suggestions and data are offered in good faith but without guarantee as conditions and methods of use of our products are beyond our control. All claims must be made within 30 days following the date of delivery. The prospective user must determine the suitability of our materials before adopting them on a commercial scale. Suggested uses of our products are not recommendations to use our products in violation of any patent or as a license under any patent of Rockland Immunochemicals, Inc. If you require a commercial license to use this material and do not have one, then return this material, unopened to: Rockland Inc., P.O. BOX 5199, Limerick, Pennsylvania, USA.
General Reference
Kong, D., L. Xu, Y. Yu, W. Zhu, D.W. Andrews, Y. Yoon, and T.H. Kuo. 2005. Regulation of Ca2+-induced permeability transition by BCL-2 is antagonized by Drp1 and hFis1. Molecular and Cellular Biochemistry. 272: 187-199. (Rabbit IgG TrueBlot, PubMed) DiPerna, G., J. Stack, A.G. Bowie, A. Boyd, G. Kotwal, Z. Zhang, S. Arvikar, E. Latz, K.A. Fitzgerald, and W.L. Marshall. 2004. Poxvirus protein N1L targets the I-kappaB Kinase complex, inhibits signaling to NF-kappaB by the Tumor Necrosis Factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by Toll-like Receptors. J. Biol. Chem. 279: 36570-36578. (Rabbit IgG TrueBlot, PubMed) Zhang, X., Y. Ozawa, H. Lee, Y. Wen, T. Tan, B. Wadzinski, and E. Seto. 2005. Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes & Development. 19: 827-839. (Rabbit IgG TrueBlot, PubMed) Lehtonen, S., E. Lehtonen, K. Kudlicka, H. Holthöfer, and M.G. Farquhar. 2004. Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin. Am J Pathol. 165:923-936. (Rabbit IgG TrueBlot, PubMed) Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. 2004. Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages. Carcinogenesis. 25: 1711-20. (Mouse IgG TrueBlot, PubMed)
Specific Reference
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Product Type
Secondary Antibodies;
Applications
FLISA, WB, IHC, IF, FC
Reacts With
Rabbit
Catalog Number
18-0216-32

Product Type
Secondary Antibodies;
Applications
FLISA, WB, IHC, IF, FC
Reacts With
Rabbit
Catalog Number
18-4416-32

Product Type
Secondary Antibodies;
Applications
FLISA, WB, IF, IP
Reacts With
Mouse
Catalog Number
18-3217-32

Product Type
Secondary Antibodies;
Applications
WB
Reacts With
Rabbit
Catalog Number
18-8816-31

Product Label

 

Description
IRDye® 800
Color
Infra-Red
Conjugation Reference
Modified from LI-COR Biosciences, Lincoln, NE
Molecular Weight
1166
Excitation Wavelength
778
Conjugation Chemistry
N-hydroxysuccinimide (NHS) ester