Tasklist
- If your AuNP colloid solution has precipitated, remake a 30:1 Au:protein (or lower Au) solution
- Prepare 500 mL 50 mM NaCl
- Prepare 1 L 50 mM Glycine buffer, pH 3.5
- As shown in prior day notebook page, I used 0.4 mL of 1 M HCl to make 100 mL of buffer
- Prepare 25 mL 1 g/L Lysozyme
- Dialyze about 4 mL of lysozyme in about 3 in of dialysis tube
- Prepare 1 3500 g/mol MWCO tubing for NaCl, Glycine, & pure water soak
- Prepare 1 15k g/mol MWCO tubing for Glycine soak
- Prepare 1 25k g/mol MWCO tubing for Glycine soak
- Dialyze 4 mL of your colloid solution in 3500 g/mol MWCO tubing for Glycine soak
- Construct pH and conductivity calibration curve for both NaCl and Glycine buffer
- For each solution, prepare 2x, 5x, 10x, 20x, 50x, 100x, 200x, and 500x dilutions
- Why don't we need anything lower than 100 μM?
- You will need 12 - 15 mL of each solution. Consider serial dilutions for each order of magnitude.
- Prepare 12 - 15 mL of 0.25 μM HCl from your HCl stock solution
- Measure the pH and conductivity of this dilution
- Use multi-well dialysis apparatus to dialyze lysozyme against:
- 50 μM, 25 μM, 2.5 μM, and 0.25 μM glycine buffer
- fill fifth well with 0.25 μM HCl
- before loading wells, you will wet a dialysis sheet and clamp it between the plastic plates
- fill one side with 1 mL of lysozyme and the other side with 1 mL of your buffer
- Analyze inside and outside dialysis membranes (pH, conductivity, Bradford) tomorrow while you wait for electrophoresis
Dialysis Protocol
- Cut about a 3" length of dialysis tubing
- 25,000 MWCO tubing is stored in an azide solution to prevent mold (use care; NaN3 is toxic)
- keep the 25,000 MWCO tubing wet to prevent pore shrinkage
- wash cut tubing with DI water, both inside and out
- 3,500 MWCO and 15,000 MWCO are dry and need to be wet prior to filling
- 25,000 MWCO needs to be rinsed to remove the sodium azide
- flatten tubing and remove as much residual water as possible with gloves fingers
- clamp one end 1/4 - 1/2" from edge
- open other end and transfer your measured protein solution inside
- carefully flatten open end and clamp, making sure no liquid escapes
- rinse with DI water, particularly the ends, which may have residual protein solution on it
- place into a 150 mL beaker (or 250 mL or 400 mL)
- measure out 100 - 200 mL of your dialyzing solution
- label beaker, cover with parafilm, and leave in your drawer overnight
- we may use the shaker on low speed to help prevent gradients during dialysis
- you should have 8 solutions when you are finished
|
Project name
Main project page
Previous entry Next entry
