Western blotting in Chlamydomonas reinhardtii

Disclaimer

DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.

Abstract

This protocols describe the steps to perform a western blot in Chlamydomonas reinhardtii cell lysate and supernatant samples.

Before start

Check antibody dilutions* Check buffers disponibility

Prepare 5% milk solution

Steps

Sample preparation - Supernatant

Centrifuge algae culture at 2000xg for 10 min1. Recover supernatant 0h 10m 0s

Sample preparation - Lysate

Centrifuge algae culture at 2000xg for 10 min1. 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. 0h 10m 0s

Sonication

Sonicate using appropriate sonication tip. Duty cycle: 0.5 | Cycle: 1.0 s | Amplitude: 20% | Duration: 30 s1. Centrifuge for 15 min at 20000xg to remove cells debries and recover soluble proteins

Quantificate soluble protein 0h 15m 0s

Gel electrophoresis | SDS-PAGE

Load 30 µg of total soluble protein (TSP) per lane in a 12% SDS-PAGE 1. 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)
Add secondary antibody if required 30