IP Detection Antibody, HRP Conjugated, SA-10060
Product Introduction
This product is a ready-to-use, HRP-conjugated secondary antibody reagent specifically designed for immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) Western blot detection. A common challenge in IP-Western blotting is the detection of target proteins at approximately 50 kDa (IgG heavy chain) and 25 kDa (IgG light chain), where denatured primary antibody fragments from the immunoprecipitation step generate strong interfering signals that obscure or co-migrate with the target protein band. This IP Detection Antibody is engineered to preferentially recognize native, non-reduced immunoglobulin, thereby eliminating or dramatically reducing the detection of denatured IgG heavy and light chain fragments on Western blots without compromising sensitivity for the target protein. The reagent is supplied as an HRP-conjugated solution for direct chemiluminescent detection, streamlining the workflow and providing a clean, high-sensitivity signal with minimal background from IP antibody interference.
Product Features
1. IP-Optimized Specificity: Engineered to eliminate or dramatically reduce detection of denatured IgG heavy chain (~50 kDa) and light chain (~25 kDa) fragments released from IP capture antibodies.
2. Does Not Detect Mouse IgG1: Specifically validated to show no cross-reactivity with mouse IgG1 isotype, making it compatible with IP experiments employing mouse IgG1 primary antibodies.
3. Ready-to-Use HRP Conjugate: Supplied as a pre-diluted HRP-conjugated solution for direct use in chemiluminescent Western blot detection, eliminating the need for secondary antibody optimization.
4. Clean Western Blot Results: Provides clear target protein bands without obscuring heavy and light chain signals, enabling unambiguous detection and quantification of co-immunoprecipitated proteins.
5. Broad Compatibility: Compatible with standard chemiluminescent (ECL) substrates and PVDF/nitrocellulose membranes.
Specifications
Size: Available in 250 µL and 500 µL
Conjugate: Horseradish Peroxidase (HRP)
Reactivity: Specifically engineered for IP detection; does not detect mouse IgG1
Recommended Dilution: 1:200 to 1:1,000
Format: Ready-to-use solution
Application: Western blot detection following immunoprecipitation and co-immunoprecipitation
Storage and Stability
Storage Conditions: Store at 4¡ãC. Do not freeze. Protect from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. After opening, use within 6 months when stored at 4¡ãC.
Protocol (For Reference Only)
Important: This reagent is specifically formulated for detection of target proteins on Western blots following IP. For best results, optimize the amount of IP antibody and IP Detection Antibody dilution for each target. The recommended working dilution is 1:200 to 1:1,000.
1. Immunoprecipitation: Perform immunoprecipitation according to standard protocols using the desired capture antibody and protein A/G agarose or magnetic beads. After washing, elute immunoprecipitated proteins by boiling in reducing SDS-PAGE sample buffer for 5 minutes.
2. SDS-PAGE and Transfer: Separate immunoprecipitated samples by SDS-PAGE alongside appropriate molecular weight markers and input controls. Transfer proteins to nitrocellulose or PVDF membrane according to standard Western blotting procedures.
3. Membrane Blocking: Block the membrane in 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature with gentle agitation. Alternative blocking buffers may be used as optimized for specific targets.
4. Primary Antibody Incubation: Incubate the membrane with primary antibody against the target protein diluted in blocking buffer or appropriate antibody dilution buffer according to the antibody manufacturer''''s recommendations. Incubate overnight at 4¡ãC or for 1-2 hours at room temperature.
5. IP Detection Antibody Incubation: After primary antibody incubation and thorough washing (3-5 washes with TBST for 5-10 minutes each), incubate the membrane with IP Detection Antibody, HRP Conjugated at a dilution of 1:200 to 1:1,000 in blocking buffer for 1 hour at room temperature with gentle agitation. For high-abundance targets, start at 1:1,000 dilution; for low-abundance targets, use 1:200 to 1:500 dilution.
6. Detection: Wash the membrane extensively (4-5 washes with TBST for 5-10 minutes each), add ECL or other HRP chemiluminescent substrate, and image using a chemiluminescence imaging system.
Precautions
1. This reagent is optimized for detection of target proteins in IP Western blots; performance may vary with different IP antibody species and isotypes. It has been validated to not detect mouse IgG1.
2. The recommended working dilution is 1:200 to 1:1,000; titrate within this range to determine the optimal dilution for each specific target and experimental system.
3. Avoid repeated pipetting from the stock bottle; aliquot working volumes and warm to room temperature before use.
4. Do not freeze; repeated freeze-thaw cycles will reduce detection activity.
5. For optimal results, use high-quality ECL substrates with appropriate sensitivity for the abundance of the target protein.
6. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the advantage of this product over standard HRP-conjugated secondary antibodies for IP-Western?
A1: Standard anti-IgG secondary antibodies detect both the native antibody used for IP and the denatured heavy and light chains released during sample boiling, producing strong interfering bands at ~50 kDa and ~25 kDa. This IP Detection Antibody is engineered to preferentially recognize native IgG, eliminating these interfering bands and providing clean detection of the target protein.
Q2: Can I use this product with mouse IgG1 capture antibodies?
A2: Yes. This product has been specifically validated to show no detection of mouse IgG1, making it fully compatible with IP experiments using mouse IgG1 isotype primary antibodies. Performance with other antibody species and isotypes should be verified experimentally.
Q3: What dilution should I use for this detection reagent?
A3: The recommended working dilution is 1:200 to 1:1,000. For high-abundance target proteins, start at 1:1,000 dilution to minimize background. For low-abundance targets or weak signals, use 1:200 to 1:500 dilution to increase sensitivity. Titration experiments are recommended for each new application.
Q4: Can I use this reagent for regular Western blot detection without IP?
A4: While this reagent is specifically engineered for IP-Western blot applications, it may be used for standard Western blot detection. However, for routine Western blotting where IP antibody interference is not a concern, standard species-specific HRP-conjugated secondary antibodies may provide equivalent or superior performance.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of detection conditions and antibody dilutions is recommended for specific experimental systems.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: SA-10060
Product Name: IP Detection Antibody, HRP Conjugated
Size: 250 µL / 500 µL
Price:
250 µL: CNY ¥1900.00 / USD $190.00 / EUR €228.00 / JPY ¥34200.00
500 µL: CNY ¥3200.00 / USD $320.00 / EUR €384.00 / JPY ¥57600.00
2023 Version
