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Protocol status: Working
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Created: April 11, 2022
Last Modified: April 11, 2022
Protocol Integer ID: 60613
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Abstract
This protocols describe the steps to perform a western blot in Chlamydomonas reinhardtii cell lysate and supernatant samples.
Guidelines
Prepare cell material by cultivating in liquid media.
Before start
Check antibody dilutions
Check buffers disponibility
Prepare 5% milk solution
Sample preparation - Supernatant
Sample preparation - Supernatant
Centrifuge algae culture at 2000xg for 10 min
Recover supernatant
00:10:00 Centrifugation time
Sample preparation - Lysate
Sample preparation - Lysate
Centrifuge algae culture at 2000xg for 10 min
Remove supernatant, and re-suspend cells in lysis buffer (50 mM Tris·HCL (pH 8.0), 0.1% Triton X-100), concentrating cells 100-fold.
00:10:00 Centrifugation time
Sonication
Sonication
Sonicate using appropriate sonication tip. Duty cycle: 0.5 | Cycle: 1.0 s | Amplitude: 20% | Duration: 30 s
Centrifuge for 15 min at 20000xg to remove cells debries and recover soluble proteins
Quantificate soluble protein
00:15:00 Centrifugation time
Gel electrophoresis | SDS-PAGE
Gel electrophoresis | SDS-PAGE
Load 30 µg of total soluble protein (TSP) per lane in a 12% SDS-PAGE
Transfer proteins to a nitrocelulose membrane
Block the membrane with 5% milk solution
Probe the desired protein with the specific antibodie, diluted in 5% milk solution
Wash 2 times with TBST (0.2 M Tris, 1.37 M NaCl, 0.1% Tween-20, pH 7.6)