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BE115+-+Lecture08 - Western blots Where the heck do I find...

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Western blots Where the heck do I find out what these blots should look like? Review articles on collagen Papers that have done similar Western blots Product information sheets for the antibodies 1
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Collagen I Made up of two different subunits α1(I) and α2(I) α1(I) is the larger of the two; both are ~95kD (but can show up higher than that on the blot) Monomers α1(I) and α2(I), dimers [α1(I)]2 and α1(I)α2(I), and trimer [α1(I)]2α2(I). 2
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Procollagen bands 3 The procollagen chains form a trimer The N and C propeptides are cleaved off, forming mature collagen Multiple mature collagens form
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Lecture 8 RNA Purification, Quantification, and Analysis; RT-PCR "Genetics explain why you look like your father, and if you don't, why you should.” - Anonymous 4
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Challenge: Record/draw/diagram in as much detail as possible the protein synthesis pathway - everything between DNA and protein. 5
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Gene-Protein Expression 6
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RNA Extraction, Quantification, and Purity Week 8 Part A 7
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Proteins vs. RNA 8 Lysis for RNA Lysis for Protein Protein Collection (lysis) Total Protein Quantification (BCA) Specific Protein Detection (Western blot) Specific Protein Quantification (Image J) mRNA Collection (lysis/purification) Total mRNA Quantification (260nm) Specific mRNA Detection (RT-PCR + agarose gel) Specific mRNA Quantification (requires special techniques like Real Time PCR or a DNA microarray)
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Western blotting vs. RT-PCR Protein Analysis: What has been made? Half-life of proteins in the body varies wildly Enzymes in the cell - hours Ornithine decarboxylase: 0.2 hours cytochrome c : 150 hrs Extracellular matrix in the body - months to years mRNA Analysis: What is being made? mRNA has a short half-life (20-60 minutes) 9
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Protein vs. mRNA Analysis How are these questions important to tissue engineering? A functional tissue needs to have the proper components to mimic necessary chemical and material properties Ideally, a tissue needs to maintain these properties (by continued protein production or other adaptation) An engineered tissue that’s perfect today may not be perfect in a week or a month unless the cells inside it are maintaining it properly 10
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Analysis of mRNA All methods of mRNA analysis require very good technique in the first step: RNA extraction and isolation Avoid contamination with Ribonucleases (RNases) - which are everywhere 11
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RNases 12 Proteins designed to hydrolyze mRNAs Seriously, they’re everywhere! Sweat Hair Skin Dust
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Protecting your RNA Samples Glassware Plastic ware Gloves Reagent bottles and tubes Solutions Temperature Designated area 13
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Glass and metal Bake at 180 oC overnight Autoclaving is not good enough 14 RNA exposed to RNase
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Plastic
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