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Western blotting is a widely used technique for detecting and quantifying specific proteins in complex mixtures. This technique involves several steps, including protein extraction, gel electrophoresis, protein transfer, and antibody detection. To perform these steps efficiently and accurately, it is essential to use high-quality buffers and stock solutions.
Western blotting begins with protein extraction, which involves lysing cells or tissues to release proteins. Lysis buffers are used for this purpose and typically contain a detergent to solubilize membrane-bound proteins. We offer a range of lysis buffers, including RIPA buffer and NP-40 buffer. RIPA buffer is a widely used buffer that includes the ionic detergent sodium deoxycholate as the active ingredient. This buffer is particularly effective for disrupting nuclear membranes in nuclear extracts. NP-40 buffer, on the other hand, contains the non-ionic detergent NP-40, which is milder than sodium deoxycholate and suitable for isolating cytoplasmic proteins.
| Recipes for RIPA buffer | Recipes for NP-40 buffer |
| 50 mM Tris-HCl (pH 8.0) 150 mM NaCl 1% IGEPAL CA-630 0.5% sodium deoxycholate 0.1% SDS Protease inhibitors | 150 mM NaCl 1.0% NP-40 (possible to substitute with 0.1% Triton X-100) 50 mM Tris-HCl, pH 8.0 Protease inhibitors |
These buffers can be stored at 4°C for several weeks, or aliquoted and stored at -20°C for up to one year.
After protein extraction, proteins are separated by gel electrophoresis. The loading buffer is added to the protein sample to denature the proteins and add tracking dye to the sample. The running buffer is utilized to create a pH gradient and facilitate protein migration through the gel. The transfer buffer transfers separated proteins from the gel to the membrane. Finally, the blocking buffer is used to block the non-specific binding of antibodies to the membrane. We offer a range of blocking buffers, including BSA (bovine serum albumin) and milk-based buffers.
| Recipes for loading buffer (PH6.8) | Recipes for transfer buffer (wet) (PH8.3) | Recipes for transfer buffer (semi-dry) |
| 4% SDS 10% 2-mercaptoethanol 20% Glycerol 0.004% bromophenol blue 0.125 M Tris-HCl | 25 mM Tris base 190 mM glycine 20% methanol | 48 mM Tris 39 mM glycine 20% methanol 0.04% SDS |
Sodium orthovanadate is a potent inhibitor of protein tyrosine phosphatases, and it is commonly used in Western blotting to enhance the signal-to-noise ratio of phospho-specific antibodies. We prepare the sodium orthovanadate formulation through the following steps:
Tris-buffered saline (TBS) and Tris-buffered saline with Tween 20 (TBST) are commonly used buffers in Western blotting. TBS is used for washing and diluting antibodies, while TBST is used for washing and diluting antibodies that require a more stringent wash.
| Recipes for TBS 10x (1 L, 20 mM Tris-HCL) | Recipes for TBST (1 L) |
| Tris 24.23 g NaCl 80.06 g 800 mL ddH2O pH to 7.5 with HCI Top up to 1 L | 100 mL TBS 10x 900 mL ddH2O 1 mL Tween 20 |
Sometimes, it is necessary to strip the membrane and re-probe it with a different antibody. This is typically done using stripping buffers that contain harsh chemicals such as SDS or urea to remove antibodies bound to the membrane. We offer both medium and harsh stripping buffers optimized for efficient antibody removal without damaging the membrane.
| Recipes for medium stripping buffer | Recipes for harsh stripping buffer |
| 15 g Glycine 1 g SDS 10 mL Tween 20 Adjust the volume to 800 mL with ultra-pure water Adjust the pH to 2.2 Top up to 1 L with distilled water | 20 mL SDS 10% 12.5 mL Tris HCl, pH 6.8, 0.5 M Adjust the volume to 100 mL with distilled water Add 0.8 mL β-mercaptoethanol |
In conclusion, the quality of the buffer and stock solutions used in Western blotting is critical for obtaining accurate and reliable results. Creative Diagnostics offers a wide range of high-quality buffers and stock solutions that are optimized for use in Western blotting. From lysis buffers to transfer buffers and stripping buffers, we have the comprehensive products you need to perform Western blotting with confidence.
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