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TrueBlot® for Generating Publication-Quality IP-Western Blotting

TrueBlot® for Generating Publication-Quality IP-Western Blots

TrueBlot® Products: Enhanced Sensitivity

Immunoprecipitation (IP) and Western Blot (WB) protocols provide highly specific results, yet can often suffer
from poor or non-specific binding, high background, and heavy/light chain interference. Rockland’s TrueBlot®
line of products consist of both TrueBlot® IP beads and TrueBlot® monoclonal secondary antibodies, which
combat these common experimental issues by providing increased sensitivity, low background, and enhanced
accuracy.

TrueBlot® Monoclonal Secondary Antibodies: Unique Detection Properties

TrueBlot® monoclonal secondary antibodies recognize the native, non-reduced form of IgG over the reduced
form of IgG in a variety of species. This unique ability makes TrueBlot® secondary antibodies the ideal solution
when performing Western blot experiments of immunoprecipitated proteins (IP–Western blot), as their use
reduces interference by the ~55 kDa heavy and ~23 kDa light chain of the immunoprecipitating antibody,
enabling unhindered detection of your blotted protein band of interest.

Features:

TrueBlot® secondary antibodies detect only native IgG, ensuring you are only detecting your target
protein
Available in HRP-conjugated forms that target mouse, rabbit, goat, or sheep IgG or fluorescent-conjugated
forms that target mouse or rabbit IgG
Easy to use–simply substitute TrueBlot® secondary antibodies for your current conventional secondary
antibodies for unparalleled clarity of results and high-quality images suitable for reproduction and
publication
Can be used in Western blot experiments that do not employ an IP step
Ideal for studying post-translational protein modifications (e.g., phosphorylation, acetylation) and protein-
protein interactions

TrueBlot® IP Beads: Exceptional Purification from Complex Samples

TrueBlot® IP beads are specifically designed, tested, and quality-controlled to provide optimal results in the
capture and purification of targets from cell lysates and serums.

Features:

Excellent binding capacities


Greater purity–stable, pre-blocked beads provide cleaner purifications
Species-specific Ig IP beads are available in an agarose or magnetic bead format, which can be paired
with TrueBlot® secondary antibodies
Variety of general use magnetic beads conjugated to Protein A, Protein G, streptavidin, or biotin
TrueBlot® Secondary Antibodies in IP–Western Blot Experiments: How do TrueBlot®
antibodies work?

Figure 1. Illustration of the detection of caspase 7 using HRP-conjugated Mouse TrueBlot® secondary
antibody vs. a conventional HRP-conjugated anti-mouse Ig secondary antibody following a typical IP
procedure

Western blots of immunoprecipitated samples can frequently result in the detection of unwanted background signals,
specifically when the same antibody is used as the capture antibody in immunoprecipitation (IP) and the primary antibody in
Western blot. Following formation of the immune complex, incubation of the complex with IP beads, and wash steps (Steps
1–3), the final steps in a conventional IP experiment typically employ a heating step in the presence of reducing/denaturing
reagents (Step 4). When the eluted IP sample is separated via SDS-PAGE, probed with the same primary antibody used in
IP, and detected with conventional secondary antibodies in Western blot, both the heavy and light chain of the IP capture
antibody can be detected (Step 5; right panel), which can obscure your protein of interest, specifically if it is similar in size to
the heavy or light chain. TrueBlot® secondary antibodies only recognize primary antibodies in their native (non-reduced)
state, eliminating interference from heavy and light chain bands (Step 6; left panel) to provide accurate, publication-quality
Western blots of immunoprecipitated samples.

TrueBlot® IP beads and TrueBlot® monoclonal secondary antibodies are available individually, in sets with the
corresponding agarose Ig IP bead, or as Western Blot kits, allowing researchers the ability to choose a TrueBlot® product to
fit all their research needs. For simple enrichment and detection of recombinant proteins, Rockland’s all-in-one TrueBlot®
IP–Western Blot kits for DYKDDDDK (FLAG®), GFP, and 6X HIS epitope tags provide the convenience of an IP–Western Blot
kit with the specificity and unique detection properties of TrueBlot® secondary antibodies. Choose from a variety of products
to easily generate reproducible, best-quality publication data using TrueBlot® products.

TrueBlot® Tutorial for IP/Western Blot

TrueBlot® Antibodies - Tutorial for IP / Weste…


Weste…

Products
Mouse TrueBlot® Immunoprecipitation Products
Rabbit TrueBlot® Immunoprecipitation Products
Goat TrueBlot® Immunoprecipitation Products
Sheep TrueBlot® Immunoprecipitation Products
TrueBlot® IP and Western Blot Kits for Epitope Tags
TrueBlot® Magnetic Beads
Fluorescent TrueBlot® Secondary Antibodies

Additional Resources
Tips for TrueBlot® Immunoprecipitation and Western Blotting
TrueBlot® Frequently Asked Questions
Agarose and Sepharose™ Conjugates

FLAG™ is a registered trademark of Sigma-Aldrich Co. LLC.

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