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Product Information

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Contents: Rabbit TrueBlot™ Western Blot Kit Catalog Number: 88-8886 Sizes: 50 ul (1000X) Storage Conditions: Upon receipt, store Rabbit IgG TrueBlot™ at less than or equal to -20°C. Store the rest of the components at 4°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.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
88-8886	Rabbit TrueBlot™ Western Blot Kit	N/A	N/A	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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The Rabbit TrueBlot^TM Western Blot Kit contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples using TrueBlot^TM second step immunoblotting reagents in conjunction with your own primary IP antibody and primary (Rabbit IgG) Western blotting antibody.

TrueBlot^TM 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-rabbit IgG reagent). Rabbit IgG TrueBlot^TM is the unique horseradish peroxidase conjugated anti-rabbit 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-rabbit IgG immunoblotting second step reagent. It is easy to generate publication-quality IP/WB data with Rabbit IgG TrueBlot^TM.

Note that there are three key procedural considerations:
1. Protein A or G should not be used for the immunoprecipitation when using Rabbit IgG TrueBlot^TM. For immunoprecipitation, anti-mouse IgG beads, anti-rat IgG beads, or anti-rabbit IgG beads should be used for mouse, rat, or rabbit immunoprecipitating antibodies, respectively.
2. Immunoprecipitate should be completely reduced.
3. Milk should be used as the blocking protein for the immunoblot.

The Rabbit TrueBlot^TM Western Blotting Kit components are sufficient for 25 miniblots.

Components:

1. Rabbit IgG TrueBlot^TM: 50 μl. An HRP-conjugated second step reagent reacting with Rabbit IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments (1000X)
2. TrueBlot^TM Enhancer Solution: 25 ml
3. TrueBlot^TM Blocker: 10 g
4. TrueBlot^TM Assay Buffer: 30 ml. 20X
5. TrueBlot^TM Substrate A: 12.5 ml
6. TrueBlot^TM Substrate B: 12.5 ml

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Applications Reported

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For research use only, not for diagnostic or therapeutic use.

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Applications Tested

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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/immunoblotting 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.

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Experimental Procedures

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Preparation of Immunoprecipitated Sample for SDS-PAGE

For optimal results, when using Rabbit IgG primary antibody, it is important to use Anti-Rabbit Ig IP Beads (Cat. No. 00-8800) for immunoprecipitation of the primary IP antibody, rather than Protein A or Protein G. The use of Protein A or Protein G is specifically not recommended; Protein A and Protein G contaminants in the immunoprecipitate bind with high affinity to blotting primary rabbit Ig, causing interfering bands at 40-60 kDa and 25-30 kDa, respectively (see Figure 1). Furthermore, it is critical to fully reduce the immunoprecipitated sample prior to loading on SDS gels. Boiling of the immunoprecipitated sample with fresh 50 mM DTT in SDS Reducing Sample Buffer should always immediately precede loading of the sample on the gel.

Figure 1: Jurkat cell lysate (0.5 ml of 1x107 Jurkat cells/ml) was incubated with rabbit anti-human Stat1 (Cat. No. 14-6011) and immunoprecipitated using Protein G, Protein A and Anti-Rabbit Ig IP Beads. Precipitate from 5x105 cells was subjected to electrophoresis, transferred to a PVDF membrane, and immunoblotted with anti-Stat1 using Rabbit IgG TrueBlot™.

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-Rabbit IgG Beads (Cat. No. 00-8800) 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-Rabbit 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-Rabbit 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-Rabbit 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. Remove membrane and soak in transfer buffer.
4. 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.]
5. 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.]
6. Incubate with immunoblotting (primary) antibody in 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer on a rocking platform at room temperature for 2 hours.
7. Wash for 1 hour at room temperature with 10 changes of TBST (approximately 6 minutes per wash).
8. Incubate with Rabbit IgG TrueBlot™ at a 1:1,000 dilution in 5% TrueBlot™ Blocker in TrueBlot™ Assay Buffer for 1 hour at room temperature.
9. Wash for 1 hour at room temperature with 10 changes of TBST (approximately 6 minutes per wash).
10. Prepare Substrate: Mix equal volumes of Substrate A and B
11. Incubate the blot in chemiluminescent-HRP substrate working solution (combined A and B) for 1-5 minutes.
12. 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.

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.

Rabbit 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.

Rabbit 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.

Rabbit IgG TrueBlot™ will detect SDS-denatured, non-reduced rabbit 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 rabbit 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.

Rabbit 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 2: Stat1 was immunoprecipitated from 0.5 ml of 1x107 Jurkat cells/ml with 3 µg rabbit anti-human Stat1. Precipitate from 5x105 cells was subjected to electrophoresis, transferred to an Immobilon membrane, and immunoblotted with anti-Stat1 using Rabbit TrueBlot™ and conventional HRP-conjugated detection. Lane 1: Detection with Rabbit IgG TrueBlot™ - note the absence of the anti-Stat1 immunoprecipitating heavy and light chains. Lane 2: re-blot of lane 1 using the conventional HRP-conjugated detection anti-rabbit polyclonal antibody - note the presence of the Stat1 immunoprecipitating heavy and light chains confirming that although the immunoprecipitating heavy and light chains were present in the sample in lane 1, Rabbit IgG TrueBlot™ detected native antibody but not the denatured heavy and light chains.

Stat1 was immunoprecipitated from 5x10⁶ Jurkat cells using 3 µg rabbit anti-Stat1 (cat# 14-6011) and 50 µl of packed Anti-Rabbit Ig IP Beads (cat# 00-8800). Immunoprecipitate was solubilized in 100 µl NuPAGE LDS sample buffer (Invitrogen, cat# NP0007) containing NuPAGE sample reducing agent (dithiothreitol; Invitrogen, cat# NP0004). Beads were pelleted and 10 µl, corresponding to antigen immunoprecipitated from 5x10⁵ cells and 0.3 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) overnight at 4°C. Stat1 was immunoblotted with 2 µg rabbit anti-Stat1 per ml in 10 ml of Buffer A containing 5% milk for 2 hours at room temperature. After washing with Buffer A the membranes were incubated with Rabbit IgG TrueBlot™ at a dilution of 1:1,000 in Buffer A containing 5% 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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Rabbit TrueBlot™ Troubleshooting Chart
Problem	Possible Cause	Solution
No Signal	Weak primary antibody NaN3 is present during HRP-substrate incubation Primary antibody is not a rabbit 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 rabbit IgG as primary antibody for Rabbit 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