This avoids the large volume of potentially hazardous hydrochloric acid that is needed to neutralize a solution of Tris base alone. The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl.Īn alternative recipe for Tris buffer combines Tris base and Tris-HCl. TBS 10x (concentrated Tris-buffered saline)Īdd distilled water to a final volume of 1 Lįor a 1x solution, mix 1 part of the 10x solution with 9 parts distilled water and adjust pH to 7.6 again. Bring up to the initial volume with waterĪvoid large changes in volume during boiling put a loose lid on the container to protect from evaporation.Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling.Prepare a 100 mM sodium orthovanadate solution with double distilled water.Failure to filter can lead to spotting, where tiny dark grains will contaminate the blot during color development.Īll procedures must be carried out under the fume hood. 3–5% milk or BSA (bovine serum albumin).For proteins >80 kDa, we recommend including SDS at a final concentration of 0.1%. Loading, running, transfer, and blocking buffers 1.0% NP-40 (possible to substitute with 0.1% Triton X-100)Ĭytoskeletal bound proteins extract buffer.The 10% sodium deoxycholate stock solution (5 g into 50 mL) must be protected from light. It can also disrupt protein-protein interactions and may, therefore, be problematic for immunoprecipitations and pull-down assays. A RIPA buffer gives low background but can denature kinases. RIPA buffer (radioimmunoprecipitation assay buffer) RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent and is particularly useful for nuclear membrane disruption for nuclear extracts. After transfer, mark the orientation of the gel on the membrane. Lysis buffers These buffers may be stored at 4☌ for several weeks or aliquoted and stored at -20☌ for up to a year.The field strength required is determined by the surface area and thickness of the gel: 0.8 mA/cm2 is a useful guide (1 h transfer). Transfer efficiency should be monitored by staining (see below). Tip: Time of transfer is dependent on the size of the proteins, percentage acrylamide, and gel thickness. For current, voltage, and transfer times specific to your apparatus, consult the manufacturer’s instructions. They can be removed by gently rolling a Pasteur pipet over each layer in the sandwich. Tip: Air bubbles may cause localized nontransfer of proteins. Tank-blotting: Avoiding air bubbles, place 4 sheets of filter paper on the fiber pad, followed by the gel, the membrane, 4 sheets of filter paper, and finally the second fiber pad.Semi-dry transfer: Avoiding air bubbles, place 4 sheets of filter paper on the cathode (negative, usually black), followed by the gel, the membrane, 4 sheets of filter paper, and finally the anode (positive, usually red).Proceed to step 4 if performing semi-dry transfer or step 5 if performing tank blotting. Soak filter paper in semi-dry or tank-blotting transfer buffer.Incubate membrane for 10 min in semi-dry or tank-blotting transfer buffer.Tip: To avoid contamination, always handle the filter paper, membrane, and gel with gloves. Cut 8 pieces of filter paper and a piece of membrane to the same size as the gel.Once transferred to the membrane, the proteins can be probed with epitope-specific antibodies or conjugates. Transfer efficiency can be checked by staining proteins on the membrane using Ponceau S (see Ponceau S staining). Results obtained with the tank-blotting method are typically better, with more efficient transfer, particularly of large proteins. With the tank-blotting method, a blotting cassette is submerged in a tank for blotting (see figure Tank- and semi-dry blotting methods).Tank blotting can be performed over extended periods since the buffer capacity is far greater than that with semi-dry transfer systems. SDS-coated, negatively charged proteins are transferred to the membrane when an electric current is applied. The membrane is placed near the anode (positively charged), and the gel is placed near the cathode (negatively charged). With the semi-dry electroblotting method, the gel and membrane are sandwiched between two stacks of filter paper that have been pre-wet with transfer buffer. Following electrophoresis, proteins in a polyacrylamide gel can be transferred to a positively charged membrane (e.g., Schleicher and Schuell BA85) in a buffer-tank–blotting apparatus or by semi-dry electroblotting as described below.
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