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Product Information

Contents: Goat TrueBlot™ Western Blot Kit
Catalog Number: 88-8884
Sizes: 50 ul (1000X)
Storage Conditions: Upon receipt, store Goat IgG Ig TrueBlot™ at -20°C and rest of the components at 4°C. Avoid 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
88-8884 Goat TrueBlot™ Western Blot Kit N/A N/A WB 

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

Description

The Goat TrueBlotTM Western Blot Kit contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples using TrueBlotTM second step immunoblotting reagents in conjunction with your own primary IP antibody and primary (Goat IgG) Western blotting antibody.

TrueBlotTM 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-Goat IgG reagent). Goat IgG TrueBlotTM is the unique horseradish peroxidase conjugated anti-Goat 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-Goat IgG immunoblotting second step reagent. It is easy to generate publication-quality IP/WB data with Goat IgG TrueBlotTM.

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 Goat TrueBlotTM Western Blot Kit components are sufficient for 25 miniblots.

Components:

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

Applications Reported

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

Applications Tested

Goat IgG TrueBlot™ is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot™ preferentially detects the non-reduced form of goat IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Goat 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. Goat IgG TrueBlot™ may also be used for detection in immunoblotting assays that do not employ immunoprecipitation.

Experimental Procedures

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-Goat IgG Beads (Cat. No. 00-8844) 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-Goat 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-Goat 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-Goat 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 Goat 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.

Goat 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.

Goat 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.

Goat IgG TrueBlot™ will detect SDS-denatured, non-reduced goat 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 goat 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.

Goat 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

Experimental Example

Figure 1: Jurkat cell lysate (0.5 ml of 1x107 Jurkat cells/ml) was incubated with goat anti-human Stat1 and immunoprecipitated using Protein G. Precipitate from 5x105 cells was subjected to electrophoresis, transferred to a PVDF membrane, and immunoblotted with anti-Stat1 using Goat IgG TrueBlot™ (lane 1) and conventional HRP conjugated anti-goat polyclonal antibody (lane 2).

Goat TrueBlot™ vs Conventional

Troubleshooting

Goat TrueBlot™ Troubleshooting Chart
Problem Possible Cause Solution
No Signal
  1. Weak primary antibody
  2. NaN3 is present during HRP-substrate incubation
  3. Primary antibody is not a goat IgG
  4. Target protein is not expressed in the sample or present at very low level
  5. Antigen is present in blocking solution
  1. Use only primary antibodies optimized for immunoblotting
  2. Incubate HRP-substrate in NaN3 free buffer
  3. Use only goat IgG as primary antibody for Goat IgG TrueBlot™
  4. Use as positive control, sample known to contain the target protein and optimize the amount of protein loaded
  5. Change blocking reagents
High background
  1. Non-optimized primary antibody
  2. Insufficient washing
  3. Membrane was allowed to dry and not re-wetted
  4. Insufficient blocking
  1. Use only primary antibodies optimized for immunoblotting
  2. Increase volume, number and duration of washes; increase salt content of the wash buffer (see Appendix)
  3. Ensure membrane is not dried during immunoblotting procedures. Immobilon-P and other PVDF membranes must be wetted in methanol and equilibrated in buffer
  4. 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
  1. Use primary antibodies optimized for immunoblotting (high signal/noise)
  2. Possible different isoforms/modifications of the protein of interest


TrueBlot™ is a Trademark of eBioscience - Patent Pending
Copyright © 2000-2008 eBioscience, Inc.
Product For Research Use Only: Not for further distribution without written consent.