Protein Characterization/ Purification. Dr. Kevin Ahern

Transcription

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.