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Part I: Preparation of Cell Lysate - Coggle Diagram
Part I: Preparation of Cell Lysate
Centrifuge bacterial culture in 50 mL aliquots at 5000g for 15 mins in a pre-chilled centrifuge.
Discard supernatant. (Pellets are frozen till ready for protein purification.)
Add PMSF (100 mM) into cell lysis buffer, washing buffer, Elution buffer 1, 2 and 3. Final concentration of PMSF in all buffer should be 1 mM
Thaw the pellet on ice.
Resuspend the pellet in 4 mL cell lysis buffer, keep cells on ice.
Sonicate cells 10 times for 10 seconds at 70% amplitude. Pause for 1min in between to prevent overheating of the protein sample. Keep samples on ice at all times.
Transfer cell lysate into two 2 mL Eppendorf tubes (4 mL in total).
Centrifuge the cell lysate at 20,000 g for 10 min. Transfer supernatant to a new tube. Keep both pellet and supernatant tubes on ice.
Observe the pellet in UV chamber. If it is still green, repeat step 1 to 8. Then, combine all supernatant.
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Part II: Column Purification
Wash the column with 10 mL water. Collect flow through in waste beaker.
Equilibrate the column with 10 mL cell lysis buffer. Collect flow through in waste beaker
Load Supernatant from step 11. Start collecting flow through into 15 mL falcon tube. Label as "Flow - Team 4"
Wash the column with 5 mL of wash buffer 2 times. (Total wash volume is 10 mL). Collect the flow through into a 15 mL collection tube. Label as "Wash - Team 4"
Elute the column with 1 mL of Elution buffer 1. Collect flow through into 2 mL collection tube. Label as "E1 - Team 4"
Elute the column with 1 mL of Elution buffer 2. Collect flow through into 2 mL collection tube. Label as "E2 - Team 4"
Elute the column with 1 mL of Elution buffer 3. Collect flow through into 2 mL collection tube. Label as "E3 - Team 4"
Repeat elution with 1 mL of Elution buffer 3. Collect the flow through into 2 mL collection tube. Label as "E3-repeat-Team 4"
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Part III: SDS-PAGE
Gather 7 protein fractions listed below. Aliquot 50 µL of each protein sample into a new tube. Add 50 µL of 2x Laemmli buffer. Heat all samples at 95 °C for 5 minutes. Store the rest of the purified protein samples in a box.
Set up PAGE gel tank. Load 5 µL of protein ladder/marker into first lane. Then load 20 µL of samples into gel according to the order in the table above. Label and store remaining samples in the box.
Run the gel at 150V for 45 min.
Remove the gel from glass plates with caution. Rinse the gel with 10 mL of water. Add 10 mL of InstantBlue to stain the gel for 20 minutes. Recycle the InstantBlue solution. Rinse the gel again with 10 mL of water.
Lab record: Take a picture of the gel.
Part IV: Fluorescence determination
(While gel running, staining and de-staining steps, measure the GFP fluorescent intensity in all the samples.) Aliquot 100 µL from each fraction of protein samples into a 96-well plate in duplicates. Add 100 µL of Lysis buffer and Elution buffer 3 in plates as well. All teams will share the plate. Please record which rows belong to your team.
Measure the GFP fluorescence using plate reader. Excitation wave length at 410 nm and Emission Wave length at 520 nm.
Lab record: Record the data and plot a bar chart using excel.