Protocol for Buffer Preparation
There are three buffers that need to be prepared for your protein purification experiment: an equilibration buffer, a wash buffer, and an elution buffer. We have optimized each buffer for the protein purification experiment. The three buffers are:
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Equilibration Buffer 50 mM NaH2PO4, pH 8.0 150 mM NaCl |
Wash Buffer 50 mM NaH2PO4, pH 8.0 150 mM NaCl 10 mM imidazole
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Elution Buffer 50 mM NaH2PO4, pH 8.0 150 mM NaCl 250 mM imidazole |
Note that all three buffers have the same concentration of sodium phosphate (provides the buffering component) and sodium chloride (used in the purification process to decrease non-specific ionic interactions between the protein of interest and the stationary phase). The buffers differ in the chemical imidazole. Note the large increase in concentration of imidazole from the wash to elution buffer. The importance of this will be explained in chapter 7. All three buffers are used during the protein purification lab (chapter 7), but we typically prepare the buffers in advance and store them in the cold room (40C fridge). It is recommended that the imidazole is added to these buffers on the day of the purification process to prevent degradation of this chemical as it is the key to proper removal of DHFR-His-6x from the stationary phase of the column. In the following protocol we describe the entire process of buffer preparation. In practice we ask our students to partially prepare the buffers with the exception of imidazole, store the buffers until the next lab, and complete the buffer preparation prior to the purification process.
In our lab course each pair of students conducts a protein purification experiment. For our setup we provide each pair of students with 10-20 mL of each buffer, but we ask a team of students to prepare 150 mL of each buffer and divide the resulting buffer between student pairs.
Please click this hyperlink to access How to Make Buffers
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Materials and Reagents
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Ensure that you properly label all prepared solutions and store them in the appropriate containers as dictated by your instructor. You will be preparing 3 buffers (150ml each):
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Equilibration Buffer 50 mM NaH2PO4, pH 8.0 150 mM NaCl |
Wash Buffer 50 mM NaH2PO4, pH 8.0 150 mM NaCl 10 mM imidazole
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Elution Buffer 50 mM NaH2PO4, pH 8.0 150 mM NaCl 250 mM imidazole |
Preparing Buffers:
1.Prior to coming to the lab calculate the amount of stock reagents you will need to prepare 150 mL of each buffer. Please record this in your lab notebook.
Note that it is not necessary for every student to make each of the buffers listed. The total amount will be dictated by the number of students and this should be discussed with the instructor at the beginning of the lab. Students can work in pairs or small groups to prepare the reagents.
2.Create proper labels for each buffer: include the full buffer name, the date it was prepared, your lab instructor name, lab section, proper handling instructions, see SDS.
3. Prepare your buffers keeping in mind all the safe handling requirements for each chemical used.
4. Once prepared, safely aliquot each buffer in the provided 50 mL conical tubes that have been properly labeled and store buffers at 40C.
Please wash your hands prior to leaving the lab.
