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Thermo Scientific Clean-Blot IP Detection Reagents are HRP and AP conjugates that are optimized for post-immunoprecipitation Western blot detection of primary antibodies without interference from denatured IP antibody fragments.
Clean-Blot IP Reagents allow trouble-free Western blot detection of target proteins following IP assays (immunoprecipitation). They function by specifically binding to functional primary antibodies (whole IgG) without also binding to fragments of the IP antibodies, which usually accompany the immunoprecipitated protein through electrophoresis and membrane transfer. The Clean-Blot IP Reagents and HRP Kit eliminate detection-interference from both heavy-chain (approx. 50kDa) and light-chain (25kDa) IgG-fragments of antibodies used for the initial immunoprecipitation assay. Clean-Blot IP products include detection reagents with alkaline phosphatase conjugate or horseradish peroxidase conjugate and the complete Clean-Blot IP Detection Kit (HRP) for chemiluminescent substrate development.
Highlights:
- Universal – bind and detect most species, IgG subclasses and isotypes of primary antibodies that are commonly used for Western blotting
- Compatible – effective with IP assays performed using Protein A, Protein G, or anti-IgG agarose beads and any blocking buffer
- Cost-effective – eliminates the cost and extra work associated with covalently immobilizing IP antibodies as a means of overcoming Western blot interference
- Flexible – available in alkaline phosphate (AP) and horseradish peroxidase (HRP) reagents for detection with chemiluminescent, fluorescent or colorimetric substrates
- Easy to use – no need to change the Western blotting protocol; simply replace conventional secondary HRP conjugate with the Clean-Blot IP Detection Reagent
- Unobstructed detection – clear Western blot results for immmunoprecipitation assays without significant interference from denatured IgG bands
Includes:
Kit contains Clean-Blot Detection Reagent (HRP), blocking buffer and Thermo Scientific Pierce ECL Substrate (chemiluminescent)
Product Details:
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Immunoprecipitation (IP) and Western blot experiments demonstrate specificity of the Thermo Scientific Clean-Blot IP Detection Reagent (HRP). The main panel (lanes 1 to 5) shows secondary detection of p53 with the Clean-Blot IP Reagent (HRP) of various control and IP conditions. The small panel (lane 6) shows detection with peroxidase-conjugated goat anti-mouse IgG (GAM-HRP).
Lane 1: A431 total cell extract expressing p53 (positive control). Lanes 2 and 3: Wash and elution fractions from no-lysate (negative control) IP and blot performed with anti-p53 primary antibody. Lanes 4 to 6: Wash and elution fractions of the complete IP experiment for p53.
As the main panel reveals, the Clean-Blot IP Reagent detects the primary anti-p53 antibody used for the Western blot but does not detect fragments of the anti-p53 antibody carried forward from the IP reaction. By contrast, the GAM-HRP (lane 6) detects both primary- and IP-components of the anti-p53 antibody.
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| Primary antibodies that are compatible with secondary detection by Thermo Scientific Clean-Blot IP Detection Reagents. Use dot blot analysis to test compatibility with specific polyclonal or monoclonal antibodies of interest. |
| Species |
Polyclonal |
Monoclonal Isotype(s) |
| Bovine |
Yes |
IgG2 |
| Goat |
Yes |
IgG2 |
| Human |
Yes |
IgG1, IgG2, IgG4 |
| Mouse |
Yes |
IgG2a, IgG2b, IgG3 |
| Rabbit |
Yes |
N/A |
| Rat |
Yes |
IgG2c |
| Sheep |
Yes |
IgG2 |
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Detect target proteins on Western blots with Thermo Scientific Clean-Blot IP Reagents without detecting endogenous IgG. Mouse liver extract (50µg) total protein was separated by SDS-PAGE, transferred to PVDF membrane and blocked with 1% milk in TBST. The membrane was probed with mouse monoclonal anti-Cdk1 (0.2µg/mL), followed by goat anti-mouse HRP (0.16µg/mL) or Clean-Blot IP Detection Reagent (HRP) (0.2µg/mL). Thermo Scientific SuperSignal West Pico Substrate (Part No. 34080) was used for final development. Liver extracts contain endogenous IgG, whose fragments are detected by anti-mouse IgG secondary antibodies (two left panels) but not by the Clean-Blot IP Detection Reagent (two right panels).
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Reveal target proteins without interference from IP antibodies. NFkB and Bax were immunoprecipitated from A549 lysate using Protein A/G agarose resin and rabbit anti-NFkB (panels A and B) and rabbit anti-Bax (Panels C and D). Fractions from each IP assay were separated by SDS-PAGE, transferred to PVDF membranes, blocked and then probed for the respective targets with the same target-specific antibodies. Panels A and C were secondarily detected with goat anti-rabbit HRP, which masked the target. Panels B and D were secondarily detected with the Clean-Blot IP Detection Reagent (HRP), unambiguously revealing the target protein without interference. All blots were developed using Thermo Scientific Pierce ECL Substrate (Part No. 32106).
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Reveal target proteins more clearly with Thermo Scientific Clean-Blot IP Reagent (AP). NFkB was immunoprecipitated from A549 lysate using Protein A/G agarose resin and rabbit anti-NFkB. Fractions from each IP assay were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked and then probed with the anti-NFkB primary antibody. Left panel blots were detected with goat anti-rabbit AP conjugate. Right panel blots were secondarily detected with the alkaline-phosphatase-conjugated Clean-Blot IP Detection Reagent (AP).
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Related Resources:
Clean-Blot IP Detection FAQ
Related Products:
Western Blotting Substrates
Blocking Buffers
Western Blot Stripping Buffers
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