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Datasheet Protocol

TrueBlot® Anti-Mouse Ig IP Agarose Beads

Goat Polyclonal

16 References
00-8811-25
2.5 mL
Suspension of agarose beads
WB, IP, SDS-PAGE, Biochemical Assay
Mouse
Goat
$220.00 /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

TrueBlot® Anti-Mouse Ig IP Agarose Beads are a suspension of activated agarose beads coupled with goat Anti-mouse IgG. It is suitable for precipitation of mouse IgGs used as the primary antibodies in immunoprecipitation assays. The beads are in suspension and will settle upon storage. Prior to use, mix the vial gently (do not vortex) to ensure delivery of proper bead volume.

Product Details

TrueBlot® Anti-Mouse Ig IP Agarose Beads - 00-8811-25
Anti-Mouse immunoglobulin Gamma, Agarose-conjugated IgG, Gt-a-Ms IgG, Goat anti-Mouse IgG, TrueBlot, TrueBlot for immunoprecipitation, IP Agarose beads for TrueBlot.
Goat
Agarose ULTRA
Polyclonal
IgG
Immunoprecipitation Kit

Target Details

Mouse
TrueBlot® Anti-Mouse Ig IP Agarose Beads has been tested in SDS-Page, immunoprecipitation, and western blot.

Application Details

IP, SDS-PAGE, WB
Biochemical Assay  - View References
Upon initial use of this product, 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, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer. Procedure: Preparation of Immunoprecipitated Sample for SDS-PAGE: 1. Preclear cell lysate: Add 50 μL of Anti-Mouse IgG Beads and 500 μL of cell lysate sample to a microcentrifuge tube and incubate on ice for 30 minutes. Spin at 10,000xg for 3 minutes and transfer the supernatant to a new microcentrifuge tube. 2. Immunoprecipitation: Add 5 μg of primary antibody to the microcentrifuge tube containing the precleared lysate. Incubate on ice for 1 hour. Add 50 μL of Anti-Mouse IgG Beads. Incubate for 1 hour on a rocking platform. Spin the microcentrifuge tube at 10,000xg for 1 minute. Remove supernatant completely and wash the (pelleted) beads 3 times with 500 μL of Lysis Buffer (50mM Tris HCl, pH 8.0; 150mM NaCl; 1% NP-40). 3. Prepare sample for SDS-PAGE: After the last wash, aspirate supernatant, and add 50 μL Laemmli Buffer (with 50 mM DTT or 2% β-mercaptoethanol, final) to bead pellet. Vortex and heat to 90-100 °C for 10 minutes. Spin at 10,000xg for 3 minutes, collect supernatant, and load onto the gel. Avoid loading Anti-Mouse Ig Beads. Note: The supernatant can be stored at -20 °C for future use. After thawing, add fresh dithiothreitol and heat as above. Centrifuge the sample at 10,000xg for 1 minute in a microcentrifuge tube to pellet any Anti-Mouse Ig Beads and immediately transfer an aliquot of the supernatant to gel wells.

Formulation

14.4 mg/cc antibody per cc agarose
0.01 M Sodium Phosphate, 0.15 M Sodium Chloride, pH 7.2
0.09% (w/v) Sodium Azide
None

Shipping & Handling

Wet Ice
Store vial at 4 °C prior to opening. DO NOT FREEZE.
Expiration date is six (6) months from date of receipt.
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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.