mouse
Articlecode: 88-8887-31
Contents: Mouse TrueBlot™ Western Blot Kit
Catalog Number: 88-8887
Sizes: 50 ul (1000X)
Storage Conditions: Upon receipt, store all components at 4°C, except the Mouse TrueBlot™, which should be stored at less than or equal to -20°C. Avoid contamination with sodium azide. This product is guaranteed for 6 months upon receipt, when handled and stored as instructed.
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Available Formats of This Product
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Reported Applications
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88-8887
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Description
The Mouse TrueBlot™ Western Blot Kit contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples using TrueBlot™ second step immunoblotting reagents in conjunction with your own primary IP antibody and primary (Mouse IgG) Western blotting antibody.
TrueBlot™ technology enables unhindered detection of protein bands of interest which would otherwise be obscured by the presence of reduced & denatured heavy and light chain immunoglobulin in the blot (as detected by the conventional immunoblotting HRP anti-mouse IgG reagent). Mouse IgG TrueBlot™ is the unique horseradish peroxidase conjugated anti-mouse IgG immunoblotting second step reagent which enables detection of immunoblotted target protein bands, without hindrance by interfering immunoglobulin heavy and light chains from your IP antibody. Use it in place of your usual HRP anti-mouse IgG immunoblotting second step reagent. It is easy to generate publication-quality IP/WB data with Mouse IgG TrueBlot™.
Note that there are two key procedural considerations:
1. Immunoprecipitate should be completely reduced.
2. Milk should be used as the blocking protein for the immunoblot.
The Mouse TrueBlot™ Western Blot Kit components are sufficient for 25 miniblots.
Components:
- Mouse IgG TrueBlotTM: 50 ìl. An HRP-conjugated second step reagent reacting with Mouse IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments (1000X)
- TrueBlotTM Enhancer Solution: 25 ml
- TrueBlotTM Blocker: 10 g
- TrueBlotTM Assay Buffer: 30 ml. 20X
- TrueBlotTM Substrate A: 12.5 ml
- TrueBlotTM Substrate B: 12.5 ml
Applications Reported
For research use only, not for diagnostic or therapeutic use. Mouse TrueBlot™ WB Kit has been reported for use in immunoblotting (WB).
Applications Tested
Mouse IgG TrueBlot™ is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot™ preferentially detects the non-reduced form of mouse IgG 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™ 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.
Related Products
Cat. 88-1488 Goat TrueBlot™ Set (includes 2.5 ml IP Beads, binds 1.0mg Ig/ml beads)
Cat. 88-1688 Rabbit TrueBlot™ Set (includes 2.5 ml IP Beads, binds 2.5mg Ig/ml beads)
Cat. 00-8811 TrueBlot™ anti-Mouse Ig IP Beads (Binds 0.4mg Ig/ml beads)
Cat. 18-8814 Goat TrueBlot™: Horseradish Peroxidase (HRP) anti-goat IgG
Cat. 18-8816 Rabbit TrueBlot™: Horseradish Peroxidase (HRP) anti-rabbit IgG
Experimental Procedures
Procedure: Preparation of Immunoprecipitated Sample for SDS-PAGE
- 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% â-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.
- 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.
- 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).
- Prepare sample for SDS-PAGE: After the last wash, aspirate supernatant, and add 100 µl Laemmli Buffer (with 50 mM DTT or 2% â-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)
- Prepare sample as indicated above and run on SDS-PAGE.
- Transfer to membrane.
- Remove membrane and soak in transfer buffer.
- Under chemical hood, place membrane in TrueBlot™ Enhancer Solution and soak for 2 minutes, then wash with TBST.
[Preparation of TBST: 25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.1% Tween 20.]
- Block with 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer. Incubate at 4°C overnight.
[Preparation of 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer: Dilute 20X TrueBlot™ Assay Buffer with dH20 to 1X. Using TrueBlot™ Blocker Powder, make a 5% (w/v) solution.]
- Incubate with immunoblotting (primary) antibody in 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer on a rocking platform at room temperature for 2 hours.
- Wash for 1 hour at room temperature with 10 changes of TBST (approximately 6 minutes per wash).
- Incubate with Mouse IgG TrueBlot™ at a 1:1,000 dilution in 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer for 1 hour at room temperature.
- Wash for 1 hour at room temperature with 10 changes of TBST (approximately 6 minutes per wash).
- Prepare Substrate: Mix equal volumes of Substrate A and B
- Incubate the blot in chemiluminescent-HRP substrate working solution (combined A and B) for 1-5 minutes.
- 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. It is recommended to block the blot in 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer overnight, which will significantly reduce the background. 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:
Experimental Example
Figure 1: Caspase-7 was immunoprecipitated from 0.5 ml of 1 x 107 Jurkat cells/ml with 5 µg mouse anti-human Caspase-7. Precipitate from 1 x 106 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.
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Caspase-7 was immunoprecipitated from 5 x 106 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 5 x 105 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.
Troubleshooting
Mouse TrueBlot™ Troubleshooting Chart
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Problem
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Possible Cause
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Solution
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No Signal
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- 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
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- 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
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High background
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- Non-optimized primary antibody
- Insufficient washing
- Membrane was allowed to dry and not re-wetted
- Insufficient blocking
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- 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.
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I see Ig in addition to my specific band of interest
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Improper sample preparation
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Follow sample preparation procedure
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I see other bands in addition to my specific band of interest
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Poor primary antibody: low signal/high noise
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- Use primary antibodies optimized for immunoblotting (high signal/noise)
- Possible different isoforms/modifications of the protein of interest
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TrueBlot™ is a Trademark of eBioscience - Patent Pending
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