Unmask your target protein in IP/Western Blots

Are antibody bands masking your target protein in IP/Western blots? Reveal your target protein using Clean-Blot IP Detection Reagent, a unique conjugate that allows clear, specific Western blot detection from IP experiments without significant interference from denatured IgG (Figure 1). Whereas conventional secondary antibodies recognize both denatured and native IgG, our new reagent binds to only native IgG (Table 1). Simply substitute your secondary antibody with Clean-Blot IP Detection Reagent for clear IP/Western blot results.

Use Clean-Blot IP Detection Reagents when Western blotting tissue extracts that contain endogenous IgGs, such as liver or spleen, for clear detection of your target protein (Figure 2). For added convenience, we have both HRP and AP (Alkaline Phosphatase) conjugates available.

Additionally, our reagent is universally compatible with primary antibodies, independent of the host species, and with IPs performed using Protein A or G Agarose Resin (Figure 3). Eliminate the need to buy separate kits based on your primary antibody species.

• Universal – Compatible with most primary antibodies independent of the host species (Table 1) 
• Compatible – Works with IPs performed using Protein A, Protein G, or anti-IgG agarose beads and any blocking buffer (e.g., milk, BSA, SuperBlock Blocking Buffer, StartingBlock Blocking Buffer)
• Cost effective – Eliminates the need to covalently crosslink IgG to a support and to purchase separate kits based on primary antibody species. Membranes can be stripped and reprobed when chemiluminescent substrate is used.
• Flexible – use HRP or AP substrate of choice, chemiluminescent, fluorescent or colorimetric
• 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 target detection – Clear IP/Western blot results without significant interference from denatured IgG bands

Figure 1: Immunoprecipitation (IP) and Western blot experiments demonstrate specificity of the Clean-Blot IP  Detection Reagent (HRP). Lane 1: A431 total cell extract expressing p53 (positive control), Lane 2 and 3: No-lysate negative control of IP wash (lane 2) and elution (lane 3) fractions, Lanes 4-6: Complete IP experiment of wash (lane 4) and elution (lane 5 and 6) fractions. Lanes 1-5 were probed with Clean-Blot IP Detection Reagent (HRP) and lane 6 was detected with GAM-HRP.

IP conditions: Anti-p53 antibody (2 µg) was added to A431 total cell extract (500 µg) expressing p53 and incubated overnight with rocking at 4 C. Protein A Agarose Resin (Product # 20333) was washed three times with binding/wash buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 5% glycerol; pH 7.5). A 50% resin slurry (100 µl) prepared with binding/wash buffer was added to each tube and rocked for 1 hour at 4°C. Non-bound protein was collected by centrifugation at 1,000 x g for 1 minute. The resin-antibody-protein complexes were washed with 500 µl of binding/wash buffer three times. P53 protein-antibody complexes were eluted by suspending the Protein A Resin in 30 µl 5X Lane Marker Reducing Sample Buffer (Product # 39000) and boiled for 5 minutes.

Western blot conditions: Proteins separated on Tris-glycine gels were transferred to PVDF membranes (Product # 88518). The membranes were blocked with 5% milk in TBST and probed with mouse anti-p53 primary antibody (BD Biosciences) (1 µg/ml) and Clean-Blot IP Detection Reagent (HRP) (0.1 µg/ml) or Goat Anti-Mouse HRP (Product # 31430, 0.16 µg/ml) as secondary conjugates. P53 protein was detected using SuperSignal West Pico Chemiluminescent Substrate (Product # 34080).

Figure 2: Easily distinguish your target protein on Western blot with Clean-Blot IP Detection Reagent (HRP). Mouse liver extract (50 µg) total protein was separated on a Bio-Rad Criterion gel, transferred to PVDF membrane and blocked with 1% milk in TBST. The membrane was probed with mouse monoclonal anti-Cdk1 (LabVision) (0.2 µg/ml) and goat anti-mouse HRP (0.16 µg/ml) or Clean-Blot IP Detection Reagent (HRP) (0.2 µg/ml).

Figure 3: Reveal your target protein with Clean-Blot IP Detection Reagent (HRP). To demonstrate unmasking of the target protein, we performed IP and Western blot experiments. 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). Panels A and C were detected with goat anti-rabbit HRP, which masked the target. Panels B and D were detected with the Clean-Blot IP Detection Reagent (HRP), revealing the target protein.

IP conditions: Four A549 cell lysate samples expressing NFkB and Bax were incubated overnight on a rocking platform at 4°C with 4 µg of rabbit anti-NFkB (Upstate) or rabbit anti-Bax (Upstate). Protein A/G Agarose (Product # 20421) was washed three times with Binding/Wash buffer (Product # 28376). A 50% Protein A/G slurry (100 l), made with Binding/Wash buffer, was added to each tube and rocked for 2 hours at room temperature. Non-bound protein was collected by centrifugation at 2,500 x g for 1 minute. The resin-antibody-protein complexes were washed with 500 µl of Binding/Wash buffer three times. Bax protein- and NFkB protein-antibody complexes were eluted by suspending the Protein A/G Agarose in 150 µl 5X Lane Marker Reducing Sample Buffer (Product # 39000) and boiled for 5 minutes.

Western blot conditions: Proteins were separated on Tris-HEPES Precise Protein Gels (Product # 25244) and transferred to PVDF membranes (Product # 88158). The membranes were blocked with StartingBlock T20 Blocking Buffer (Product # 37543) and probed with rabbit anti-NFkB or rabbit anti-Bax primary antibodies (1 µg/ml, Upstate) and Pierce Native IgG Detection Reagent (HRP) at 0.4 µg/ml (Panels B and D) or Goat Anti-Rabbit HRP (Product # 31460) at 0.16 µg/ml (Panels A and C). NFkB and Bax were detected using Pierce ECL Chemiluminescent Substrate (Product # 32106).

Figure 4: Reveal your target protein with Clean-Blot IP Reagent (AP). NFkB was immunoprecipitated from A549 lysate using Protein A/G Agarose Resin and rabbit anti-NFkB (Panels A and B). Panel A was detected with Clean-Blot IP Detection Reagent (AP) revealing NFkB and Panel B was detected with Goat Anti-Rabbit AP (Product # 31340), which masked the protein.

IP conditions: The IP was performed by combining A549 cell lysate and Anti-NFkB antibody (rabbit polyclonal, Upstate, 5 ug) and incubating at room temperature for one hour. Twenty µL of 50% slurry Immobilized Protein A/G (Product # 20421) was washed with PBS buffer before adding to each IP reaction. The IP reaction was incubated for 1 hour at RT. The flow-through was collected after centrifugation at 2000 x g for 1 minute. After washing (4x with PBS), 100 µL of 5X Reducing sample buffer (50% glycerol, 0.3M Tris, pH 6.8 5% SDS, 100 mM DTT) was added to each reaction and incubated for 5 minutes at 95°C in a Reacti-Therm. Ten µL from each reaction was loaded onto a 4-20% Precise gel (Product #25224).

Western blot conditions: Proteins were separated on Tris-HEPES Precise Protein Gels (Product # 25244) and transferred to nitrocellulose membranes (Product # 77012). The membranes were blocked with StartingBlock T20 Blocking Buffer (Product # 37543) and probed with rabbit anti-NFkB primary antibody (0.9 µg/ml, Upstate) and Clean-Blot Detection Reagent (AP) at 0.1 µg/ml (Panel A) or Goat Anti-Rabbit AP (Product # 31340) at 0.05 µg/ml (Panel B). NFkB was detected using Lumi-Phos WB Chemiluminescent Substrate (Product # 34150).

Ordering Information

Certificate of Analysis   Product Instructions   MSDS

Buy Product # Description Pkg. Size Files Price Add 21230 Clean-Blot IP Detection Reagent (HRP) sufficient reagent for approximately 100 Western blots 2.5 ml $150.00 Add 21232 Clean-Blot IP Detection Kit (HRP) sufficient reagents for approximately 2000 cm2 of membrane Kit contains: Pierce Native IgG Detection Reagent (HRP) 2.5 ml StartingBlock T20 (TBS) Blocking Buffer 1L Pierce ECL Detection Reagent 1, Peroxide Solution 125 ml Pierce ECL Detection Reagent 2, Luminol Enhancer Solution 125 ml Kit $275.00 Add 21233 Clean-Blot IP Reagent (AP) sufficient reagent for approximately 100 Western blots 2.5 ml $150.00