WDR70 Mouse mAb Protocol: IHC, WB & IP Applications

WDR70 Mouse mAb is a murine monoclonal antibody designed for the specific detection of human WDR70 protein in western blot (WB), immunohistochemistry (IHC), and immunoprecipitation (IP) experiments.

Product SKU

Ab349685

Antibody Type

Mouse Monoclonal Antibody (mAb)

Target Protein

WDR70 (WD repeat-containing protein 70)

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP)

Host Species

Mouse

Reference Catalog

absea #KC-36160

Overview of WDR70 and Antibody Specifications

WDR70 (WD repeat-containing protein 70) is a member of the WD-repeat protein family, characterized by tandem WD40 motifs that form a beta-propeller scaffold mediating protein–protein interactions. WDR70 has been implicated in chromatin remodeling and DNA damage response pathways, making it a relevant target in cell biology and cancer research.

WDR70 Mouse mAb (Aladdin SKU: Ab349685; reference absea #KC-36160) is a murine monoclonal antibody raised against a specific epitope of the human WDR70 protein. As a monoclonal antibody, it offers high batch-to-batch consistency and defined epitope specificity, which are critical for reproducible experimental results across western blotting, immunohistochemistry, and immunoprecipitation workflows.

Sample Preparation

Cell and Tissue Lysates (for WB and IP):

1. Harvest cells by scraping or centrifugation and wash twice with ice-cold PBS.
2. Lyse cells in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail. Incubate on ice for 30 minutes with occasional vortexing.
3. Centrifuge at 12,000 × g for 15 minutes at 4°C. Collect the supernatant.
4. Determine protein concentration using a BCA or Bradford assay.
5. Aliquot and store lysates at −80°C until use.

Tissue Sections (for IHC):

1. Fix tissues in 10% neutral-buffered formalin and embed in paraffin (FFPE), or prepare frozen sections as appropriate.
2. For FFPE sections, perform antigen retrieval by heating slides in citrate buffer (10 mM, pH 6.0) at 95–100°C for 10–20 minutes, then allow to cool gradually.
3. Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes.

Note: Specific validated dilutions and buffers for WDR70 Mouse mAb (Ab349685) should be confirmed in the product datasheet. Flag for review.

Step-by-Step Protocol by Application

Western Blot (WB)

1. Separate 20–40 µg of total protein by SDS-PAGE on an appropriate percentage gel.
2. Transfer proteins to a PVDF or nitrocellulose membrane.
3. Block membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature.
4. Incubate with WDR70 Mouse mAb (Ab349685) at the recommended dilution (see product datasheet) in blocking buffer overnight at 4°C.
5. Wash 3 × 10 minutes with TBST.
6. Incubate with an appropriate HRP-conjugated anti-mouse secondary antibody for 1 hour at room temperature.
7. Wash 3 × 10 minutes with TBST, then detect signal using an ECL substrate system.

Immunohistochemistry (IHC)

1. Following antigen retrieval and peroxidase blocking, rinse sections with PBS.
2. Block non-specific binding with 5–10% normal serum (from secondary antibody host species) for 30 minutes at room temperature.
3. Apply WDR70 Mouse mAb (Ab349685) at the recommended dilution and incubate overnight at 4°C in a humidified chamber.
4. Rinse sections with PBS (3 × 5 minutes) and apply a biotinylated anti-mouse secondary antibody or polymer-based detection system.
5. Develop with DAB substrate, counterstain with hematoxylin, dehydrate, and mount.

Immunoprecipitation (IP)

1. Pre-clear lysate (500–1000 µg total protein) with protein A/G agarose beads for 1 hour at 4°C on a rocker.
2. Transfer supernatant to a fresh tube and add WDR70 Mouse mAb (Ab349685) at the recommended amount. Incubate overnight at 4°C with rocking.
3. Add protein A/G agarose beads and incubate for a further 2–4 hours at 4°C.
4. Wash beads 3–5 times with lysis buffer. Elute bound proteins with 2× SDS sample buffer by boiling at 95°C for 5 minutes.
5. Analyze eluate by SDS-PAGE and western blot.

Note: Optimal antibody concentrations and dilutions must be determined empirically or confirmed from the product datasheet. Flag for review.

Troubleshooting

The following troubleshooting guide addresses common issues encountered when using WDR70 Mouse mAb (Ab349685):

Problem	Possible Cause	Suggested Solution
No signal in WB	Insufficient protein loaded; antibody dilution too high; inadequate transfer	Increase protein load; optimize antibody concentration; verify transfer efficiency with Ponceau S staining
High background in WB	Blocking insufficient; secondary antibody cross-reactivity; antibody concentration too high	Extend blocking time or switch blocking reagent; increase wash stringency; titrate primary antibody
Non-specific bands	Antibody concentration too high; cross-reactivity with related WD-repeat proteins	Titrate antibody; include a negative control; confirm band size against expected molecular weight
Weak or no IHC staining	Inadequate antigen retrieval; over-fixation of tissue	Optimize antigen retrieval conditions; use fresh tissue fixation protocols
High background in IHC	Insufficient blocking; endogenous peroxidase not quenched	Extend blocking step; ensure complete peroxidase quenching

For application-specific recommended dilutions and positive control tissues, always consult the current product datasheet for Ab349685.

Storage, Handling and Safety

Storage: Store WDR70 Mouse mAb (Ab349685) according to the conditions specified on the product datasheet and vial label. Monoclonal antibodies are generally stored at −20°C for long-term storage. Avoid repeated freeze-thaw cycles, which can reduce antibody activity. Aliquoting upon first use is recommended.

Handling: Thaw on ice or at 4°C before use. Briefly centrifuge the vial after thawing to collect the contents. Keep on ice during experimental use. Do not expose to prolonged light, especially if the antibody is conjugated to a fluorophore.

Safety: Follow standard laboratory safety practices when handling antibody preparations. Consult the Safety Data Sheet (SDS) supplied with the product for hazard information. Dispose of biological materials in accordance with local institutional and regulatory guidelines.

Note: Specific storage temperature, buffer composition, and expiry date must be confirmed from the product datasheet for Ab349685. Flag for review.

Frequently asked questions

What applications is WDR70 Mouse mAb (Ab349685) validated for?

WDR70 Mouse mAb (Ab349685) is intended for use in western blot (WB), immunohistochemistry (IHC), and immunoprecipitation (IP) applications. For confirmed validated applications and species reactivity, please refer to the current product datasheet.

What is the recommended dilution for WDR70 Mouse mAb in western blot?

Optimal dilution ranges for WDR70 Mouse mAb (Ab349685) should be obtained from the product datasheet, as validated dilutions are determined empirically during product development. A typical starting range for monoclonal antibodies in WB is 1:500–1:2000, but this must be confirmed for this specific product.

How should I store WDR70 Mouse mAb to maintain its activity?

Monoclonal antibodies are generally stored at −20°C for long-term use. To preserve activity, avoid repeated freeze-thaw cycles by aliquoting the antibody upon first use. Refer to the Ab349685 product datasheet for the exact recommended storage conditions and expiry information.

What positive control should I use when testing WDR70 Mouse mAb?

An appropriate positive control for WDR70 Mouse mAb would be a cell line or tissue known to express WDR70. Specific positive control recommendations, including validated cell lines or tissue types, should be confirmed from the Ab349685 product datasheet.

Can WDR70 Mouse mAb be used with non-human samples?

Species reactivity depends on sequence homology between the immunogen epitope and the target species' WDR70 protein. Confirmed species reactivity for Ab349685 should be verified in the product datasheet, as cross-reactivity with non-human species has not been specified in the available source material.

References

1. absea Product Reference: KC-36160 — WDR70 Mouse Monoclonal Antibody. Absea Biotechnology Ltd.
2. Aladdin Scientific Product Listing: Ab349685 — WDR70 Mouse mAb. Aladdin Scientific.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.