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Contents: Mouse TrueBlot™ ULTRA: Horseradish Peroxidase (HRP) anti-mouse IgG Catalog Number: 18-8817 Storage Conditions: Upon receipt, store at less than or equal to -20°C. Avoid freeze/thaw cycles and contamination with sodium azide. This product is guaranteed for 6 months upon receipt, when handled and stored as instructed. Prices for This Product* Cat. No. Size Price Qty Action 18-8817-30 20 ul (1000X) $40 18-8817-31 50 ul (1000X) $85 18-8817-33 200 ul (1000X) $185 *International customers: Please contact your distributor for region specific pricing.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
18-8817	Mouse TrueBlot™ ULTRA: Horseradish Peroxidase (HRP) anti-mouse IgG	N/A	N/A	IP  WB

Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq.

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Description

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Mouse IgG TrueBlot^TM ULTRA is a unique horseradish peroxidase (HRP) conjugated anti-mouse IgG immunoblotting (second step) reagent, and has more HRP molecules per antibody than the original Mouse IgG TrueBlot^TM (cat. 18-8877). Mouse IgG TrueBlot^TM ULTRA 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 data with Mouse IgG TrueBlot^TM ULTRA, simply substitute the conventional HRPO anti-Mouse IgG blotting reagent with Mouse IgG TrueBlot^TM ULTRA and follow the prescribed protocol for sample preparation and immunoblotting.

Note that there are two key procedural considerations:
1. complete reduction of immunoprecipitate
2. effective blocking of the Western blot using milk as blocking protein

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Applications Reported

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For research use only, not for diagnostic or therapeutic use. Mouse TrueBlot™ ULTRA has been reported for use in immunoprecipitation, and immunoblotting (WB).

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Applications Tested

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Mouse IgG TrueBlot™ ULTRA is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. Mouse IgG TrueBlot™ ULTRA preferentially detects the non-reduced form of mouse IgG (IgG1, IgG2a, IgG2b, IgG3) over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Mouse IgG TrueBlot™ ULTRA 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/immunoblotting applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions.

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References

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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-κB Kinase complex, inhibits signaling to NF-κB by the Tumor Necrosis Factor superfamily of receptors, and inhibits NF-κB 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)

Okoshi Y, Tahara-Hanaoka S, Nakahashi C, Honda S, Miyamoto A, Kojima H, Nagasawa T, Shibuya K, Shibuya A. 2005. Requirement of the tyrosines at residues 258 and 270 of MAIR-I in inhibitory effect on degranulation from basophilic leukemia RBL-2H3. Int Immunol. 17(1):65-72. (Mouse IgG TrueBlot, PubMed)

Murray, J., M.F. Marusich, R.A. Capaldi, and R. Aggeler. 2004. Focused proteomics: Monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain. Electrophoresis. 25:2520-2525. (Mouse IgG TrueBlot, PubMed)

Hamdane, M., A. Bretteville, A. Sambo, K. Schindowski, S. Begard, A. Delacourte, P. Bertrand, and L. Buee. 2005. p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death. Journal of Cell Science. 118: 1291-1298. (Rabbit and Mouse IgG TrueBlot, PubMed)

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Experimental Procedures

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Procedure: Preparation of Immunoprecipitated Sample for SDS-PAGE

1. Immediately before use, make 2x SDS Reducing Sample Buffer by adding 1M DTT to 2x SDS Sample Buffer to a final concentration of 50 mM DTT. NuPAGE or standard Laemmli buffer may also be used with the addition of reducing agent (50 mM DTT or 2% β-mercaptoethanol, final).
   
   2x SDS Reducing Sample Buffer (containing 50 mM DTT)
   
   • 950 ml of 2x SDS sample buffer
   • 50 ml of 1M DTT
   • Use within 1 hour and discard remainder.
   2x SDS Sample Buffer
   
   • 6% SDS
   • 25 mM Tris base pHed to 6.5 with HCl
   • 10% glycerol
   • Bromphenol blue
   • Can be stored long term at -20°C and for up to 1 month at room temperature.
   1M DTT
   
   • Can be made fresh or can be stored as aliquots at -20°C for 6 months or at 4°C for 2 weeks. Avoid repeated freeze thaws.
   
   
2. Preclear cell lysate: Add 50 µl of Anti-Mouse IgG Beads (Cat. No. 00-8811) and 500 µl of cell lysate sample to a microcentrifuge tube and incubate on ice for 30 minutes. Spin at 10,000 x g for 3 minutes and transfer the supernatant to a new microcentrifuge tube.
3. 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 10,000 x g 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).
4. Prepare sample for SDS-PAGE: After the last wash, aspirate supernatant, and add 100 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 10,000 x g 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 10,000 x g 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.
   

Procedure: Immunoblotting (Western Blotting, WB)

1. Prepare sample as indicated above and run on SDS-PAGE.
2. Transfer to membrane.
3. Block with 5% milk in Buffer A (25 mm Tris-HCl, pH 7.3, 0.15 M NaCl, 0.1% Tween-20) overnight at 4°C on a rocking platform. Note: Milk solution should be stored at 4°C short term or -20°C for long term.
4. Incubate with immunoblotting (primary) antibody in 5% milk in Buffer A on a rocking platform at room temperature for 2 hours or overnight at 4°C.
5. Wash for 1 hour at room temperature with 10 changes of Buffer A (approximately 6 minutes per wash).
6. Incubate with Mouse IgG TrueBlot™ at a 1:1,000 dilution in 5% milk in Buffer A for 1 hour at room temperature.
7. Wash for 1 hour at room temperature with 10 changes of Buffer A (approximately 6 minutes per wash).
8. Develop the blot using a chemiluminescent-HRP substrate according to instructions provided by the manufacturer.
9. Expose the membrane to X-ray film for the desired time. A 1 minute exposure is a suggested starting time and the exposure time can be shortened or lengthened as desired. Extended exposure times (> 5 minutes) are not recommended.

Other Procedural Notes

1.  Titration of primary IP antibody.

Titration of the immunoprecipitating antibody is recommended (e.g., 1-5 µg / 10⁷ cells / 1 ml lysate). Typically, 2 µg is a sufficient amount of antibody to maximally immunoprecipitate most antigens in 1 ml of extract from 1 x 10⁷ cells. Using as little IP antibody as possible minimizes potential contamination of SDS reduced sample with nonreduced immunoprecipitating antibody light chain. 5 µg of immunoprecipitating antibody per ml of extract from 0.5 x 10⁷ - 1.0 x 10⁷ cells yields sample loads of 0.5-1.5 µg immunoprecipitating antibody and precipitates from1 x 10⁶- 3 x 10⁶ cells.

Mouse IgG TrueBlot™ will not improve the specificity of immunoblotting antibody. If the immunoblotting antibody cross-reacts with proteins other than the target, the bound immunoblotting antibody will be detected by TrueBlot™.

2. Blocking the immunoblot.

Mouse IgG TrueBlot™ has been extensively tested in blotting conditions that employ 5% (w/v) nonfat milk powder for blocking, 5% (w/v) nonfat milk powder for immunoblotting with primary antibody, and 5% (w/v) nonfat milk powder for incubation with TrueBlot™. Blocking solution is made from 25 mM Tris, pH 7.3, 0.15 M NaCl, and 0.1% Tween-20. Always readjust pH to 7.3 after addition of nonfat milk powder, which acidifies the buffer. Use of other proteins for blocking and incubation with immunoblotting antibody and TrueBlot™ is not recommended. The use of BSA for blocking is specifically not recommended.

3. Positive control.

Mouse IgG TrueBlot™ will detect SDS-denatured, non-reduced mouse IgG. A 20 ng sample of non-reduced, immunoprecipitating antibody can be included in the immunoblot as a positive control to ensure positive performance of TrueBlot™.

4. Negative control.

Sample containing 0.5-2.0 µg of reduced mouse IgG (prepared and run immediately as described in Sample Preparation) can be included as a negative control to ensure that TrueBlot™ does not detect the heavy and light chain of the immunoprecipitating antibody.

Other potential controls include omitting the cell extract during the IP, omitting the IP antibody during the IP, or omitting the immunoblotting antibody.

5. SDS-PAGE.

Mouse IgG TrueBlot™ has been tested in procedures that employed anti-oxidant in the running buffer, staining of the membrane with Ponceau S Solution (Sigma) followed by destaining with 20% acetic acid/30% methanol, and air drying the membrane for 1 hour at room temperature after transfer and then rewetting in methanol and equilibration in Buffer A (described in Immunoblotting Protocol) before blocking. These modifications of the protocol gave results identical to the Immunoblotting Protocol. The Immunoblotting Protocol has not included these variations.

Additional information:

For updated information on this product.
http://www.ebioscience.com
General guidelines for immunoprecipitation and immunoblotting protocols can be found at:
http://www.ebioscience.com/ebioscience/appls/IP.htm
http://www.ebioscience.com/ebioscience/appls/WB.htm

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Experimental Example

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Figure 1:  Caspase-7 was immunoprecipitated from 0.5 ml of 1x107 Jurkat cells/ml with 5 µg mouse anti-human Caspase-7. Precipitate from 1x106 cells was subjected to electrophoresis, transferred to an Immobilon membrane, and immunoblotted with anti-caspase-7 using conventional HRP-conjugated detection anti-mouse polyclonal antibody or Mouse IgG TrueBlot™. Lane 1: detection with Mouse IgG TrueBlot™ - note the absence of the anti-caspase-7 immunoprecipitating heavy and light chains. Lane 2: detection with conventional HRP-conjugated anti-mouse antibody - note the detection of the anti-caspase-7 immunoprecipitating heavy and light chains. Lane 3: re-blot of lane 1 using the conventional HRP-conjugated detection anti-mouse polyclonal antibody - note the presence of the caspase-7 immunoprecipitating heavy and light chains confirming that although the immunoprecipitating heavy and light chains were present in the sample in lane 1, Mouse IgG TrueBlot™ detected native antibody but not the denatured heavy and light chains.

Caspase-7 was immunoprecipitated from 5x10⁶ Jurkat cells using 5 µg mouse anti-caspase-7 (Cat. No. 14-6697) and 50 µl of packed protein-G beads (Amersham). Immunoprecipitate was solubilized in 100 µl NuPAGE LDS sample buffer (Invitrogen) containing NuPAGE samples reducing agent (dithiothreitol) (Invitrogen). Beads were pelleted and 10 µl, corresponding to antigen immunoprecipitated from 5x10⁵ cells and 0.5 µg immunoprecipitating antibody, were electrophoresed on 4-12% minigels (Invitrogen) and transferred to Immobilon-P membranes (Millipore). Membranes were blocked with 5% lowfat dry milk in Buffer A (25 mM Tris, pH 7.3, 0.15 M NaCl, 0.1% Tween-20) for 1 hour at room temperature. Caspase-7 was immunoblotted with 2 µg anti-caspase-7 per ml in 10 ml of Buffer A containing 2% milk for 2 hours at room temperature. After washing with Buffer A the membranes were incubated with Mouse IgG TrueBlot™ at a dilution of 1:1,000 in Buffer A containing 2% milk for 1 hour at room temperature. Membranes were washed with Buffer A and developed with ECL reagent (Amersham) and exposed to film.

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Troubleshooting

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Mouse TrueBlot™ Troubleshooting Chart
Problem	Possible Cause	Solution
No Signal	Weak primary antibody NaN3 is present during HRP-substrate incubation Primary antibody is not a mouse IgG Target protein is not expressed in the sample or present at very low level Antigen is present in blocking solution	Use only primary antibodies optimized for immunoblotting Incubate HRP-substrate in NaN3 free buffer Use only mouse IgG as primary antibody for Mouse IgG TrueBlot™ Use as positive control, sample known to contain the target protein and optimize the amount of protein loaded Change blocking reagents
High background	Non-optimized primary antibody Insufficient washing Membrane was allowed to dry and not re-wetted Insufficient blocking	Use only primary antibodies optimized for immunoblotting Increase volume, number and duration of washes; increase salt content of the wash buffer (see Appendix) Ensure membrane is not dried during immunoblotting procedures. Immobilon-P and other PVDF membranes must be wetted in methanol and equilibrated in buffer 5% (w/v) nonfat dry milk is the best blocking agent.  BSA is specifically not recommended.
I see Ig in addition to my specific band of interest	Improper sample preparation	Follow sample preparation procedure
I see other bands in addition to my specific band of interest	Poor primary antibody: low signal/high noise	Use primary antibodies optimized for immunoblotting (high signal/noise) Possible different isoforms/modifications of the protein of interest

TrueBlot™ is a Trademark of eBioscience - Patent Pending