WB not working well? Faint or missing bands? Your sample extraction method might be the issue!
Using collagen, which is difficult to extract, as an example, let's tackle the WB experiment together!
Collagen is insoluble in cold water and organic solvents. Therefore, conventional tissue/cell lysis methods are not efficient for collagen extraction. Here, we recommend using the heat SDS extraction method for collagen, which is convenient, fast, and highly efficient.
▇ Sample Extraction Recommendation – SDS Extraction Method
1. For animal sampling, complete the procedure within 5 min. After dissection, gently rinse the tissue with pre-cooled PBS (G2156) to remove surface blood until clean. Use filter paper to absorb excess liquid, then immediately place the sample into an EP tube or cryogenic vials (CV-3) and store at -80℃. For transportation, quickly transfer the sample to a low-temperature environment (dry ice) for preservation and shipping.
2. Cut a 100mg tissue sample into pieces and place it into a 2mL grinding tube (HT-200-M). Add 10× volume (1mL) of RIPA (strong) lysis buffer (G2002), pre-mixed with Cocktail (G2006), phosphoprotease inhibitor (G2007), and PMSF (G2008). Add 2 pcs of 4mm zirconia grinding beads. Since collagen is temperature-sensitive, extraction should be performed on ice at 4℃ to prevent collagen degradation due to the unwinding of its triple helix.
3. Place the grinding tube into the low-temperature homogenizer (SWE-FP) for processing. Set the grinding parameters to 4℃, 70Hz, for 60s. For tougher samples like skin, repeat the homogenization multiple times at 20s intervals to achieve optimal results.
4. After grinding, immediately add 10% SDS solution (G2055) to adjust the final SDS concentration to 4% based on the homogenate volume. Mix thoroughly, then heat in a dry bath (SMB-H) at 90°C for 15min.
5. Centrifuge the lysate at 12,000 rpm at room temperature for 15min. Collect the supernatant, reserving a portion for BCA Protein Quantitative Detection Kit (G2026) to evaluate protein extraction efficiency.
6. Mix the protein supernatant with 5× loading buffer (G2075) at a 4:1 ratio and denature at 95°C for 15min in a dry bath.
(The product catalog numbers used above are all from the Servicebio brand.)
▇ Recommended Experimental Conditions
1. Gel Preparation: Use the 15-minute One-step Fastcast Colorful Acrylamide Kit (6%) (G2175).
2. Electrophoresis: Use standard electrophoresis buffer (G2144). Run the stacking gel at 90V for 30min until it enters the resolving gel. Adjust the voltage to 120V and continue electrophoresis until bromophenol blue reaches 1 cm from the bottom of the glass plate, then stop electrophoresis.
3. Transfer: Use fast transfer buffer (G2148) for quick transfer without ice bath, at 300 mA for 40-60 minutes.
4. Blocking: Block with 5% skimmed milk (GC310001), incubate at room temperature for 1 hour.
5. Primary Antibody Incubation: Incubate overnight at 4°C on a shaker (DS-3D100).
6. Secondary Antibody Incubation: Incubate at room temperature on a shaker (DS-3D100) for 1 hour.
7. Chemiluminescence: Use ECL detection reagent (G2161, a combination set with different sensitivities suitable for all protein concentrations from low to high, reducing overexposure and improving success rates).
8. Chemiluminescence Imaging: Perform chemiluminescence imaging (SCG-W3000 is recommended, capable of exposing both weak and strong signals in one exposure).
(The product catalog numbers used above are all from the Servicebio brand.)
▇ Comparison of SDS Extraction Method vs. Standard Extraction Method Results
1. Cat. No.: GB114197
Lane 1: Mouse skin tissue lysate
Predicted band size: 139 kDa
Lane 2: Mouse skin tissue lysate (SDS extraction method)
Detected band size: 120-140 kDa
Lane 3: Rat skin tissue lysate
Lane 4: Rat skin tissue lysate (SDS extraction method)
▇ Comparison of SDS Extraction Method vs. Standard Extraction Method Results
2. Cat. No.: GB11022
Lane 1: Mouse skin tissue lysate (RIPA extraction method)
Lane 2: Rat skin tissue lysate (RIPA extraction method)
Lane 3: Mouse skin tissue lysate (SDS extraction method)
Lane 4: Rat skin tissue lysate (SDS extraction method)
Loading Control Antibodies are Essential for WB Experiments.
