Struggling with XPO1 antibody band in western blot?

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Hi does anyone have any experience with XPO1 antibody? My current antibody for XPO1 is from Cell Signalling Technology and I am not really getting good bands in western blot. Does anyone have any recommendation for this?

Zein Al-Abideen Douba

If you're encountering issues with detecting XPO1 (Exportin-1) in your Western blot using the Cell Signaling Technology (CST) antibody (Catalog #46249), here are some troubleshooting steps and considerations to help improve your results:

Troubleshooting Tips for XPO1 Western Blot

Antibody Dilution and Incubation Conditions Dilution: CST recommends using a 1:1,000 dilution for Western blot applications. However, depending on your sample type and expression levels, you might need to optimize this dilution. Incubation Time: Incubate the primary antibody overnight at 4°C with gentle agitation to enhance binding specificity and sensitivity. Fresh Antibody Preparation: Always prepare antibody dilutions fresh to avoid degradation and microbial contamination, which can affect performance Cell Signaling Technology.

Sample Preparation Protein Extraction: Ensure efficient lysis of your cells or tissue to obtain a clear protein extract. Use appropriate lysis buffers and include protease inhibitors to prevent protein degradation. Protein Quantification: Accurately quantify protein concentrations using methods like the BCA assay to ensure equal loading across lanes. Loading Control: Include a loading control, such as β-actin or GAPDH, to verify equal protein loading and transfer efficiency.

Gel Electrophoresis and Transfer Gel Percentage: Use a 7.5–10% SDS-PAGE gel, as XPO1 has a molecular weight of approximately 123 kDa, which is suitable for detection on these gels. Transfer Efficiency: Confirm efficient transfer of proteins to the membrane. Perform a Ponceau S stain to check for uniform transfer before proceeding with antibody incubation.

Blocking and Washing Blocking: Block the membrane with 5% non-fat dry milk or BSA in TBST to prevent non-specific binding. Washing: Wash the membrane thoroughly with TBST (3× 5 minutes) to remove unbound antibodies and reduce background noise.

Secondary Antibody and Detection Secondary Antibody: Use an HRP-conjugated secondary antibody that is compatible with your primary antibody's host species. Detection Method: Employ chemiluminescent substrates like ECL to visualize the bands. Ensure that the exposure time is optimized to prevent overexposure or underexposure.

Alternative XPO1 Antibodies

If issues persist with the CST antibody, consider trying antibodies from other reputable suppliers:

Proteintech: Offers a CRM1 antibody (Catalog #66763-1-Ig) suitable for Western blot applications.
Aviva Systems Biology: Provides an XPO1 antibody (Catalog #OACA10444) with applications in Western blot, immunohistochemistry, and immunofluorescence.

These alternatives may offer different sensitivities or specificities that could be beneficial depending on your experimental setup.

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