Western Blotting is a cornerstone technique in molecular biology, enabling researchers to detect and analyze specific proteins in complex samples. This protocol provides a detailed, step-by-step guide for preparing cell and tissue lysates, running SDS-PAGE, transferring proteins to membranes, and performing both indirect and direct immunoblotting. It ensures reliable protein detection for a wide range of studies, from cancer biology to protein expression analysis, and is supported by abinScience’s high-quality antibodies and reagents.
■ Sample Preparation
-
Monolayer Cells:
-
Grow cells to subconfluency in a 100 mm culture dish. Remove the culture medium and rinse the cell monolayer once with room-temperature PBS to remove residual media. Place the dish on ice, then add 0.6 ml of ice-cold RIPA buffer to the cells and gently rock for 15 minutes at 4°C to lyse the cells. Use a cell scraper to dislodge adherent cells, transferring the lysate to a microcentrifuge tube. Rinse the dish with an additional 0.3 ml of RIPA buffer and combine with the original lysate for maximum recovery. Optionally, add protease inhibitors like PMSF and/or shear DNA by passing the lysate through a 21-gauge needle to reduce viscosity. Incubate on ice for 30–60 minutes to ensure complete lysis. Centrifuge at 10,000 × g for 10 minutes at 4°C, collecting the supernatant as the whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA buffer, centrifuge again, and combine the supernatants.
-
Suspension Cells:
-
Harvest approximately 2 × 10⁷ cells by low-speed centrifugation (e.g., 200 × g for 5 minutes). Wash the cell pellet with PBS to remove contaminants, then centrifuge again to collect the cells. Resuspend the pellet in 1 ml of ice-cold RIPA buffer, ensuring freshly added protease and/or phosphatase inhibitors are included to preserve protein integrity. Incubate on ice for 30 minutes, with occasional gentle pipetting to facilitate lysis. Optionally, shear DNA using a 21-gauge needle, dounce homogenization, or sonication to reduce sample viscosity, taking care to avoid heating the lysate. Centrifuge at 10,000 × g for 10 minutes at 4°C, and collect the supernatant as the whole cell lysate for downstream analysis.
-
Tissue Samples:
-
Accurately weigh and finely mince the tissue using a clean razor blade to ensure efficient lysis. For frozen samples, thinly slice and thaw in RIPA buffer containing protease and/or phosphatase inhibitors to protect proteins during extraction. Use approximately 3 ml of buffer per gram of tissue to achieve optimal lysis. Homogenize the tissue using a dounce homogenizer or sonicator, keeping samples at 4°C to prevent protein degradation. Optionally, add PMSF for additional protease inhibition. Incubate on ice for 30 minutes to allow complete lysis. Centrifuge at 10,000 × g for 10 minutes at 4°C to separate the lysate, and clarify by centrifuging again if needed. The supernatant serves as the total protein extract for Western Blot analysis.
■ SDS-PAGE and Protein Transfer
-
Procedure:
-
Mix the prepared sample with an equal volume of 2× sample buffer to denature proteins, then boil at 95–100°C for 2–3 minutes to ensure complete denaturation. Load 40–60 µg of whole cell lysate per lane on an SDS-PAGE gel, adjusting the amount based on the sample type and protein abundance. Include a molecular weight marker in a separate lane for reference. Run the SDS-PAGE gel under standard electrophoresis conditions, typically at 100–150 V, until the dye front reaches the bottom. Transfer the separated proteins to a PVDF or nitrocellulose membrane using either wet or semi-dry electrotransfer methods, following the equipment manufacturer’s protocol to ensure efficient protein transfer.
■ Immunoblotting – Indirect Detection
-
Blocking:
-
Block nonspecific binding sites on the membrane by incubating it in 5% non-fat milk or BSA in TBS or TBST for 30–60 minutes at room temperature. Alternatively, block overnight at 4°C for enhanced specificity, ensuring the membrane is fully submerged in the blocking solution.
■ Immunoblotting – Direct Detection
-
Direct ECL:
-
Block the membrane as described in the indirect method to reduce non-specific binding. Incubate the membrane with an HRP-conjugated primary antibody at 0.5–2 µg/ml in blocking buffer for 2 hours at room temperature. Wash the membrane thoroughly to remove unbound antibody, then detect using ECL reagent and image with a suitable system.
This Western Blot protocol ensures reliable protein detection for a wide range of research applications. abinScience provides high-quality antibodies and reagents to support your experiments, ensuring reproducibility and accuracy. For more protocols, products, or technical support, visit abinScience’s official website or contact our team at support@abinscience.com.
