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Rabbit TrueBlot® Set (IP Agarose beads)

7 References
88-1688-31
1 Set
WB, IP
Rabbit
$292.00 /Per Item
Availability: Ships next business day
Shipping info:

$50.00 to US & $70.00 to Canada for most products. Final costs are calculated at checkout.

Background

Rabbit IgG TrueBlot® is a unique horseradish peroxidase conjugated Anti-Rabbit IgG monoclonal secondary antibody. 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/Western Blot data with Rabbit IgG TrueBlot®, simply substitute the conventional HRP Anti-Rabbit IgG blotting reagent with Rabbit IgG TrueBlot® 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 blot applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions. Rabbit IgG TrueBlot may also be used for detection in immunoblotting assays that do not employ immunoprecipitation.

Product Details

Rabbit TrueBlot® Set (with IP Agarose beads) - 88-1688-31
Anti-Rabbit Immunoglobulin Gamma, Agarose-conjugated IgG, Gt-a-Rb IgG, Goat-anti-Rabbit IgG, TrueBlot for immunoprecipitation, IP Agarose beads for TrueBlot, HRP, HRP TrueBlot ULTRA, Peroxidase TrueBlot, TrueBlot for IP/WB, TrueBlot for western blotting
Peroxidase (Horseradish) ULTRA
Monoclonal
IgG
Immunoprecipitation Kit

Target Details

Rabbit
Rabbit TrueBlot® Antibody Peroxidase 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.

Application Details

IP, WB
Rabbit IgG TrueBlot® is provided as 1000X solution. 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 will yield enough reagent for 20 blots. Note that there are three key procedural considerations: 1. Protein A or G should not be used for the immunoprecipitation. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands. 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. BLOTTO/Milk should be used as the blocking protein for the immunoblot. Rabbit TrueBlot Set Components: 1. Rabbit IgG TrueBlot® HRP-conjugated monoclonal secondary antibody reacting with rabbit IgGs for optimal signal detection in immunoprecipitation/immunoblot experiments. 2. Anti-Rabbit Ig IP Beads: 2.5 mL. Binds 1 mg Ig/mL beads. 3. Western blot incubation tray. Special Notes: Upon initial use of the IP beads, we recommend that the vial be inverted several times to get the beads into suspension. We recommend using a large bore pipet to pipet up the liquid for use. For storage of the opened vial of beads, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer. All recommended dilutions for listed applications are intended as an initial recommendation, specific conditions for each protein and antibody combination should be specifically optimized by the end user.

Formulation

Liquid (sterile filtered)
0.01 M Sodium Phosphate, 0.15 M Sodium Chloride, pH 7.2
0.1 mg/ml Bovine Serum Albumin (BSA) - IgG and Protease free, 50% (v/v) Glycerol

Shipping & Handling

Wet Ice
Store TrueBlot® Anti-Rabbit Ig IP beads (00-8800-25) at 2-8 °C and Rabbit TrueBlot® (18-8816-31) at -20 °C. This product is guaranteed for 6 months upon receipt, when handled and stored as instructed.
Expiration date is six (6) months from date of receipt.
(). ZBP1 sequesters cGAS in the cytoplasm and sustains type I interferon responses to mitochondrial DNA. bioRxiv Preprint
Applications
IP, Co-IP
(). Identification of Nucleolin as a Novel AEG-1-Interacting Protein in Breast Cancer via Interactome Profiling. Cancers (Basel).
Applications
IP, Co-IP
(). Oncogenic ZEB2 activation drives sensitivity toward KDM1A inhibition in T-cell acute lymphoblastic leukemia. Blood.
Applications
IP, Co-IP
(). High-resolution time-lapse imaging and automated analysis of microtubule dynamics in living human umbilical vein endothelial cells. J Vis Exp.
Applications
IP, Co-IP
(). Epo-induced erythroid maturation is dependent on Plcγ1 signaling. Cell Death & Differentiation
Applications
IP, Co-IP; WB, IB, PCA
(). A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity. Hum Mol Genet.
Applications
IP, Co-IP
(). Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells. Skeletal Muscle
Applications
WB, IB, PCA

What is Rockland’s TrueBlot® product line?

Rockland’s TrueBlot® product line is designed to solve common experimental problems when performing immunoprecipitation/co-immunoprecipitation experiments and Western blots of immunoprecipitated samples (IP–Western blot). The product line consists of TrueBlot® monoclonal secondary antibodies and TrueBlot® IP beads.

What is the advantage of TrueBlot® secondaries over regular secondary antibodies?

TrueBlot® antibodies are specific to the whole IgG molecule and will not bind heavy or light chains. This is useful for binding target proteins with an expected MW near 25 kDa (light chains) and 50 kDa (heavy chains).

Why does it appear that the TrueBlot® antibody is binding heavy/light chains in my sample?

The sample must be fully reduced to eliminate cross-reactivity with heavy and light chains. Any reactivity to heavy and light chains could be due to incomplete reduction of your sample. Please be sure to optimize reducing conditions for your sample type.

How can I ensure my samples are fully reduced?

Samples can be fully reduced by heating at 90-100°C for 10 minutes in sample buffer containing reducing agent (for example, DTT to a final concentration of 50 mM or add β-Mercaptoethanol to a final concentration of 2% (v/v)).

With which species/isotypes are TrueBlot® secondary antibodies reactive?

TrueBlot® secondary antibodies are reactive with the IgG of their respective species. For example, if using a primary antibody raised in mouse (mouse host), use TrueBlot® anti-mouse Ig secondary antibodies for detection.

Do TrueBlot® secondary antibodies detect IgM?

TrueBlot® secondary antibodies have not been shown to detect IgM.

Do I need to preclear my lysate before the immunoprecipitation step?

Samples that have many other proteins present (such as lysates) may require preclearing to prevent interference in later IP and WB procedures. Recombinant protein samples require pre-clearing more often than serum samples.

How can I enrich/concentrate my lysate for a low expression of a protein of interest?

The immunoprecipitation procedure can be repeated several times to yield a more concentrated protein solution.

What is the recommended lysis buffer for TrueBlot® products?

The optimal lysis buffer will depend on your sample type. See protocol for buffer recommendations. Generally, 1X RIPA is used to lyse samples.

My target protein has the same MW as a heavy/light chain. How can I be sure that the band is the target protein and not a heavy/light chain?

Be sure to include a positive control for the primary antibody in your experiment and check that the sample is fully reduced.

What is the average size of TrueBlot® magnetic/agarose beads?

The beads are roughly 0.5 μm in diameter.

Why can’t I use Protein A or Protein G beads for the IP step in IP–Western blot when using rabbit species?

Using Protein A or Protein G beads with rabbit species results in contaminated bands. For best results when using rabbit species, use Rockland’s TrueBlot® Rabbit Ig IP beads, which are available in either a magnetic or agarose format. Please see individual TrueBlot® Rabbit product pages for additional details.

Can I use Protein A or Protein G beads for the IP step in IP–Western blot when using mouse, goat, or sheep species?

Yes. If you are using Protein A or Protein G beads with mouse, goat, or sheep species, we recommend the following: Determine the compatibility of Protein A or Protein G with your species and subtype, do not use excessive amounts of slurry, and choose the appropriate elution conditions. In some instances, harsher elution conditions can cause stripping of the subunits of Protein A or Protein G from the bead support and result in non-specific bands. For best results, we recommend using TrueBlot® Ig IP beads, which are available in either agarose or magnetic format.

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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.