Protein Characterization/ Purification. Dr. Kevin Ahern
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1 Protein Characterization/ Purification Dr. Kevin Ahern
2 Protein Purification Applications of Biochemistry Knowledge
3 Protein Purification Applications of Biochemistry Knowledge Opening Cells
4 Protein Purification Applications of Biochemistry Knowledge Opening Cells Centrifugation
5 Protein Purification Applications of Biochemistry Knowledge Opening Cells Centrifugation Fractionation
6 Dialysis Applications of Biochemistry Knowledge
7 Dialysis Applications of Biochemistry Knowledge Separates salts from proteins
8 Chromatography (Column) Applications of Biochemistry Knowledge
9 Chromatography (Column) Applications of Biochemistry Knowledge Separation based on charge - Ion Exchange
10 Chromatography (Column) Applications of Biochemistry Knowledge Separation based on charge - Ion Exchange Separation based on size - Size Exclusion / Gel Filtration
11 Chromatography (Column) Applications of Biochemistry Knowledge Separation based on charge - Ion Exchange Separation based on size - Size Exclusion / Gel Filtration Separation based on affinity - Affinity Chromatography
12 Chromatography (Column) Applications of Biochemistry Knowledge Separation based on charge - Ion Exchange Separation based on size - Size Exclusion / Gel Filtration Separation based on affinity - Affinity Chromatography Separation based on polarity - Reverse Phase Chromatography
13 Ion Exchange Chromatography Cation exchange chromatography (+ sticks) Anion exchange chromatography (- sticks)
14 Cation Exchange Chromatography
15 Size Exclusion / Gel Filtration Chromatography
16 Size Exclusion / Gel Filtration Chromatography
17 Affinity Chromatography
18 Reverse Phase HPLC Chromatography
19 Reverse Phase HPLC Chromatography Columns have non-polar packing material Non-polar materials interact more with column than polar materials The most polar materials will elute first. The most non-polar materials will elute last.
20 Agarose Gel Electrophoresis
21 Agarose Gel Electrophoresis
22 Agarose Gel Electrophoresis Mesh-like support
23 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative)
24 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells
25 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support
26 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio
27 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest
28 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest
29 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest Loading Wells
30 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest Loading Wells Largest
31 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest Loading Wells Largest Smallest
32 Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest
33 Polyacrylamide Gel Electrophoresis
34 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores
35 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein)
36 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells
37 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support
38 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio
39 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest
40 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest
41 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest Largest
42 Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest Largest Smallest
43 Isoelectric Focusing
44 Isoelectric Focusing
45 Isoelectric Focusing
46 Metabolic Melodies
47 The Proteins Marching One by One To the tune of "The Ants Go Marching One by One Lyrics by Tari Tan Oh there's a method you should know that's very huge It's spinning round and round inside the centrifuge The supernatant, pellet too You choose the one that's right for you And from there we pu-ri-fy What's inside To size exclude filtration is the way to go The beads have pores small proteins can go in you know The largest ones, they come out fast The smallest ones eluting last And the proteins purified By their size Electrons power gel e-lec-tro-pho-re-sis The protein is denatured thanks to SDS Proteins in a minus state Get sorted by atomic weight Smaller ones in speedy mode To the anode Ion exchange is special chromatography To switch cations, you must have a minus bead Upon this bead, the proteins bind They're positive, not any kind And the others wash right through Out to you Oh my this song has given you a mighty list Perhaps we'll just skip over ol' dialysis So study HPL and C If you have questions, talk to me You will get through protein hell You'll do well.
48 Proteomics - 2D Gel Electrophoresis
49 Proteomics - 2D Gel Electrophoresis In Proteomics, Researchers Aim to Quantitate All of the Proteins Made in Cell/Tissue
50 Proteomics - 2D Gel Electrophoresis In Proteomics, Researchers Aim to Quantitate All of the Proteins Made in Cell/Tissue 2-D Gel Electrophoresis is One Way to Do This Analysis
51 Proteomics - 2D Gel Electrophoresis Add Protein Mixture to Polyelectrolyte Column
52 Proteomics - 2D Gel Electrophoresis Apply Electrical Current
53 Proteomics - 2D Gel Electrophoresis Apply Electrical Current High pi Proteins Separate According to pi Values Low pi
54 Proteomics - 2D Gel Electrophoresis Rotate Apply to Gel
55 Proteomics - 2D Gel Electrophoresis Rotate Apply to Gel Add SDS Separate By Size on Polyacrylamide Gel
56 Proteomics - 2D Gel Electrophoresis Rotate Apply to Gel Add SDS Separated By Charge/pI Separate By Size on Polyacrylamide Gel
57 2- D Gel Electrophoresis
58 2- D Gel Electrophoresis
59 2- D Gel Electrophoresis
60 Proteomics - 2D Gel Electrophoresis Separated By Charge/pI Separated By Size
61 Proteomics - 2D Gel Electrophoresis Separated By Charge/pI Separated By Size Each Spot Corresponds to a Unique Protein
62 Proteomics - 2D Gel Electrophoresis Separated By Charge/pI Separated By Size Each Spot Corresponds to a Unique Protein The Intensity of Each Spot is a Measure of the Amount of Protein Present
63 2- D Gel Electrophoresis
64 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous
65 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange
66 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue
67 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue
68 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue Orange - Proteins in a Healthy Cell, But Not a Cancer Cell
69 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue Orange - Proteins in a Healthy Cell, But Not a Cancer Cell Blue - Proteins in a Cancer Cell, But Not a Healthy Cell
70 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue Orange - Proteins in a Healthy Cell, But Not a Cancer Cell Blue - Proteins in a Cancer Cell, But Not a Healthy Cell
71 Biotechnology Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue Orange - Proteins in a Healthy Cell, But Not a Cancer Cell Blue - Proteins in a Cancer Cell, But Not a Healthy Cell Black - Proteins Equally Abundant in Both Cells
72 Microarray Analysis Microarray Analysis Allows a Researcher to Measure the Quantity of every mrna of Interest Made in a Cell/Tissue
73 Microarray Analysis Chemically Synthesize DNA Corresponding to an mrna
74 Microarray Analysis Chemically Synthesize DNA Corresponding to an mrna Bond Thousands of Copies of That DNA to a Spot on a Slide
75 Microarray Analysis Repeat for Every mrna of an Organism
76 Microarray Analysis Repeat for Every mrna of an Organism One Spot Per mrna
77 Microarray Analysis Gene #1
78 Microarray Analysis Gene #2 Gene #1
79 Microarray Analysis Gene #2 Gene #3 Gene #1
80 Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous
81 Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous
82 Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous Isolate All mrnas from Each
83 Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous Copy Each mrna Using Reverse Transcriptase to Make cdna Copies of Each
84 Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous Add Fluorescent Green Tag to Normal Cell cdnas
85 Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous Add Fluorescent Green Tag to Normal Cell cdnas Add Fluorescent Red Tag to Cancer Cell mrnas
86 Microarray Analysis Mix cdna Samples
87 Microarray Analysis Mix cdna Samples
88 Microarray Analysis Mix cdna Samples
89 Microarray Analysis Mix cdna Samples Pour Mixture Onto Slide
90 Microarray Analysis Mix cdna Samples Pour Mixture Onto Slide Allow Hybridization to Occur
91 Microarray Analysis Mix cdna Samples Pour Mixture Onto Slide Allow Hybridization to Occur Wash Unhybridized Samples Away
92 Microarray Analysis
93 Microarray Analysis Intensity of Color Measures Amount of mrna
94 Microarray Analysis Intensity of Color Measures Amount of mrna Shade of Color Measures Relative Expression Between Cell Types
95 Microarray Analysis Intensity of Color Measures Amount of mrna Shade of Color Measures Relative Expression Between Cell Types Bright Green - Abundant In Healthy Cells, Not in Cancer Cells
96 Microarray Analysis Intensity of Color Measures Amount of mrna Shade of Color Measures Relative Expression Between Cell Types Bright Green - Abundant In Healthy Cells, Not in Cancer Cells Bright Red - Abundant In Cancer Cells, Not in Healthy Cells
97 Microarray Analysis Intensity of Color Measures Amount of mrna Shade of Color Measures Relative Expression Between Cell Types Bright Green - Abundant In Healthy Cells, Not in Cancer Cells Bright Red - Abundant In Cancer Cells, Not in Healthy Cells Bright Yellow - Abundant In Both Cells Types
98 Microarray Analysis Intensity of Color Measures Amount of mrna Shade of Color Measures Relative Expression Between Cell Types Bright Green - Abundant In Healthy Cells, Not in Cancer Cells Bright Red - Abundant In Cancer Cells, Not in Healthy Cells Black - Absent in Both Cells Types Bright Yellow - Abundant In Both Cells Types
99 Microarray Analysis
100 Western Blotting Useful for identifying proteins in a gel
101 Western Blotting Proteins Must be Transferred from Gel to a Membrane
102 Western Blotting Detection uses Labeled Antibody Specific to Protein of Interest
103 Metabolic Melodies
104 I ve Just Run a Gel (to the tune of "I've Just Seen a Face") by Kevin Ahern and Indira Rajagopal I ve just run a gel. I do not think it went too well I may have used a bit much SDS. The stacker s looking like a mess. It s true Oh now what will I do? The protein sample s my last one. To purify it was not fun I spent three weekends working late. The middle lanes aren t looking great. I m screwed Good God what will I do? Crawling. I m almost bawling The boss is calling to follow through I just loaded all I ve got to make this final western blot My fingers are both crossed for sure I hope my protein product s pure. I do Then my thesis is through
105 I ve just run a gel. I do not think it went too well I may have used a bit much SDS. The stacker s looking like a mess. It s true Oh now what will I do? The protein sample s my last one. To purify it was not fun I spent three weekends working late. The middle lanes aren t looking great. I m screwed Good God what will I do? Crawling. I m almost bawling The boss is calling to follow through I just loaded all I ve got to make this final western blot My fingers are both crossed for sure I hope my protein product s pure. I do Then my thesis is through I ve Just Run a Gel (to the tune of "I've Just Seen a Face") by Kevin Ahern and Indira Rajagopal Hating. All of the waiting I m contemplating what I should do Staining. My eyes are straining There s no complaining. I say wahoo Cuz it has the band I need I ll go and have it scanned to speed The writing of my thesis and Proceed onto the post-doct ral plan Oh that will be so grand Pieces make up my thesis. No more phoresis. The promised land. Writing so unexciting. But no more biting. My nails again. Writing is coinciding. With reference citing. I m at the end.
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