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Western Blotting for Chondroitin Sulfate Proteoglycans

This protocol was developed by Dr. Panpan Yu, Associate Professor, Guangdong–Hongkong–Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China when she was a Research Fellow in the lab.  Please contact her at: yupanpan21@gmail.com for further help.

  1. Protein samples are separated by SDS-PAGE on a 6% polyacrylamide gel under reducing conditions.

  2. Electroblot the SDS-PAGE-separated samples onto the PVDF membrane using semi-dry or wet transfer device. (Note: Reduce the methanol concentration in transfer buffer to 10 % instead of 20%, to facilitate transfer of high molecular weight proteins such as CSPGs).

  3. Block the membrane in blocking buffer (5% milk in PBS-Tween) at room temperature for 1 h or overnight at 4°C on the shaker.

  4. Incubate the membrane overnight at 4°C on the shaker with primary antibody CS-56 (1:500, Sigma) diluted in 5% milk in PBS-Tween; incubate for additional 2 h the following day at room temperature.

  5. Wash the membrane 3 times for 10 min each with PBS-Tween.

  6. Incubate the membrane with HRP-conjugated goat anti-mouse IgM secondary antibody at room temperature for 35 min.

  7. Wash the membrane 6 times for 5 min each with PBS-Tween.

  8. Detect the signals using chemiluminescent methods. (Note: use high sensitive chemiluminescent reagents)

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