sds page and western blot protocol...1 sds page and western blot protocol laboratory - day 1...
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SDS PAGE and Western BlotProtocol
Laboratory - Day 1
• Bring in fish samples.• Analyze relatedness among samples using
the phylogenic charts.• Complete Laboratory Day 1 protocol
– Laemmli sample buffer• Solubilize the proteins in the fish muscle samples• Tris buffer, SDS, bromophenol blue, and glycerol.
– Molecular weight marker– Actin and myosin standard
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Lab - Day 1: Extract and Denature FishProteins
Lab Day 2: Gel loading, running andstaining
• Defrost the protein extracts.• Prepare the electrophoresis chamber• Prepare the pre-cast gel and lock it into the
electrophoresis chamber.• Load and run the gel.
– 1x TGS buffer Tris-glycine-SDS• Stain the gel
– Coomassie stain
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Lab Day 2: Prepare Chamber
Lab Day 2: Prepare Chamber
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Lab Day 2: Gel loading, running andstaining
Lab Day 2: Gel loading, running andstaining
**Change volume to 5ul for all samples
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Lab Day 2: Stain the gel
**DO NOT DISCARD THE RUNNING BUFFER!
LAB DAY 3: Western Blotting
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LAB DAY 3: Western Blotting
LAB DAY 3: Western Blotting
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LAB DAY 3: Western Blotting
LAB DAY 3: Western Blotting
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LAB DAY 4: Immunodetectionfor Myosin Light Chains
LAB DAY 4: Immunodetectionfor Myosin Light Chains
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LAB DAY 4: Immunodetection forMyosin Light Chains
Lab Day 5: Analysis• Take a picture of your gel.• Analyze your results.
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