SDS PAGE and Western Blot Protocol · 1 SDS PAGE and Western Blot Protocol Laboratory - Day 1...
9
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
Transcript of SDS PAGE and Western Blot Protocol · 1 SDS PAGE and Western Blot Protocol Laboratory - Day 1...
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
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
3
Lab Day 2: Prepare Chamber
Lab Day 2: Prepare Chamber
4
Lab Day 2: Gel loading, running andstaining
Lab Day 2: Gel loading, running andstaining
**Change volume to 5ul for all samples
5
Lab Day 2: Stain the gel
**DO NOT DISCARD THE RUNNING BUFFER!
LAB DAY 3: Western Blotting
6
LAB DAY 3: Western Blotting
LAB DAY 3: Western Blotting
7
LAB DAY 3: Western Blotting
LAB DAY 3: Western Blotting
8
LAB DAY 4: Immunodetectionfor Myosin Light Chains
LAB DAY 4: Immunodetectionfor Myosin Light Chains
9
LAB DAY 4: Immunodetection forMyosin Light Chains
Lab Day 5: Analysis• Take a picture of your gel.• Analyze your results.
![Quantification of 2D Gel Western Blot Images from … · Quantification of 2D Gel Western Blot Images from Human Tumor Samples Nancy Kendrick, ... [1,2] Anaplastic Lymphoma ... Genomic](https://static.fdocuments.net/doc/165x107/5b61d6747f8b9a54488cc28b/quantification-of-2d-gel-western-blot-images-from-quantification-of-2d-gel-western.jpg)
