sds page and western blot protocol...1 sds page and western blot protocol laboratory - day 1...

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1 SDS PAGE and Western Blot Protocol 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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Page 1: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

1

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

Page 2: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

2

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

Page 3: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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Lab Day 2: Prepare Chamber

Lab Day 2: Prepare Chamber

Page 4: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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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

Page 5: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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Lab Day 2: Stain the gel

**DO NOT DISCARD THE RUNNING BUFFER!

LAB DAY 3: Western Blotting

Page 6: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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LAB DAY 3: Western Blotting

LAB DAY 3: Western Blotting

Page 7: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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LAB DAY 3: Western Blotting

LAB DAY 3: Western Blotting

Page 8: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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LAB DAY 4: Immunodetectionfor Myosin Light Chains

LAB DAY 4: Immunodetectionfor Myosin Light Chains

Page 9: SDS PAGE and Western Blot Protocol...1 SDS PAGE and Western Blot Protocol Laboratory - Day 1 •Bring in fish samples. •Analyze relatedness among samples using the phylogenic charts

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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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