Scientific Methods (I)
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Scientific Methods (I) Jau-Song Yu ( 余余余 ) Department of Cell and Molecular Biology CGU 1
description
Scientific Methods (I). Jau-Song Yu ( 余兆松 ) Department of Cell and Molecular Biology CGU. 1. What Are Scientific Methods?. 2. What Are Scientific Methods?. The way scientists discover the truth. 3. What kind of truths you want to discover?. 4. What kind of truths you want to - PowerPoint PPT Presentation
Transcript of Scientific Methods (I)
Scientific Methods (I)
Jau-Song Yu ( 余兆松 )
Department of Cell and Molecular BiologyCGU
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What Are Scientific Methods?
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What Are Scientific Methods?
The way scientists discover the truth
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What kind of truths you want to discover?
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What kind of truths you want to discover?
Curiosity
Used to keep watching on curious things
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What kind of truths you want to discover?
Curiosity
Used to keep watching on curious things
How to learn scientific methods?
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To Learn Scientific Methods ----
To be an apprentice ( 學徒 ) in a lab under the instruction of a teacher or teachers*
*What can we learn from our supervisor(s)?
Other sources for learning scientific methods??*
*Can we learn by ourselves??? When, Where and How????
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What can we learn from our supervisor ?
Basic experimental techniques (1-10 or more) (enough or not enough?) (instruction manual)
The topic(s) and the problem(s) that the supervisor is interested in (how important and what is the interesting point) (‘good’ or ‘good enough’ science, the wider-range of impact)
The way about the formation of the question (hypothesis) that the supervisor wants to address (to solve a important question remained unanswered or just for fun……)
The thinking logic that the supervisor has applied to solve the problem (read a lot of papers and then point out 1-10 methods or just by intuition……)
The way (experimental techniques and attitude) to solve the problem (right methods? Insist or compromise when encounter difficulty? how detail he(she) wants to ask into? how does he(she) explain the data? the attitude about the “truth”)
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Basic experimental techniques (1-10 or more) (enough or not enough?) (instruction manual)
The principle behind the technique and its limitation --- ( 君子務本 , 本立道生 ) determination of protein concentration measurement of cell viability, mobility, ROS…. The “feeling” about a series of steps in performing one experiment The ability to foresee results --- Expectation
The “control” group --- how many controls should be done in one experiment?
Authority (it must be….. 想當然耳…… .)
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Determination of protein concentration
Warburg-Christian method --- A280 for Trp, Tyr
Lowry method --- folin phenyl reagent
Modified Lowry method
Bradford method --- Protein dye-binding
BCA assay kit --- bicinchoninic acid/Cu2+
Gel-based method
(1 mg/ml protein >>> A280 ~0.4-1.5; parvalbumins = 0, lysozyme = 2.65)
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J Biol Chem. 1951 Nov;193(1):265-75.
MCDONALD CE, CHEN LL. Anal Biochem. 10:175-7. (1965) THE LOWRY MODIFICATION OF THE FOLIN REAGENT FOR DETERMINATION OF PROTEINASE ACTIVITY.
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Measurement of cell viability
Trypan Blue dye exclusion
MTT assay
Other methods ??
Measurement of cell motility
Wound healing assayTranswell assaySingle cell or a collection of cells? Other methods ??
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Cell Death Differ. 2009 Aug;16(8):1093-107. Epub 2009 Apr 17.Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnóczky G, Hardwick JM, Hengartner MO, Ichijo H, Jäättelä M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nuñez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JH, Puthalakath H, Rabinovich GA, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Scorrano L, Simon HU, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G. INSERM, U848, Villejuif, France.
Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.
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Autophagy. 2008 Feb 16;4(2):151-75. Epub 2007 Nov 21.Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X, Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT, Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu CT, Chung J, Clarke PG, Clark RS, Clarke SG, Clavé C, Cleveland JL, Codogno P, Colombo MI, Coto-Montes A, Cregg JM, Cuervo AM, Debnath J, Demarchi F, Dennis PB, Dennis PA, Deretic V, Devenish RJ, Di Sano F, Dice JF, Difiglia M, Dinesh-Kumar S, Distelhorst CW, Djavaheri-Mergny M, Dorsey FC, Dröge W, Dron M, Dunn WA Jr, Duszenko M, Eissa NT, Elazar Z, Esclatine A, Eskelinen EL, Fésüs L, Finley KD, Fuentes JM, Fueyo J, Fujisaki K, Galliot B, Gao FB, Gewirtz DA, Gibson SB, Gohla A, Goldberg AL, Gonzalez R, González-Estévez C, Gorski S, Gottlieb RA, Häussinger D, He YW, Heidenreich K, Hill JA, Høyer-Hansen M, Hu X, Huang WP, Iwasaki A, Jäättelä M, Jackson WT, Jiang X, Jin S, Johansen T, Jung JU, Kadowaki M, Kang C, Kelekar A, Kessel DH, Kiel JA, Kim HP, Kimchi A, Kinsella TJ, Kiselyov K, Kitamoto K, Knecht E, Komatsu M, Kominami E, Kondo S, Kovács AL, Kroemer G, Kuan CY, Kumar R, Kundu M, Landry J, Laporte M, Le W, Lei HY, Lenardo MJ, Levine B, Lieberman A, Lim KL, Lin FC, Liou W, Liu LF, Lopez-Berestein G, López-Otín C, Lu B, Macleod KF, Malorni W, Martinet W, Matsuoka K, Mautner J, Meijer AJ, Meléndez A, Michels P, Miotto G, Mistiaen WP, Mizushima N, Mograbi B, Monastyrska I, Moore MN, Moreira PI, Moriyasu Y, Motyl T, Münz C, Murphy LO, Naqvi NI, Neufeld TP, Nishino I, Nixon RA, Noda T, Nürnberg B, Ogawa M, Oleinick NL, Olsen LJ, Ozpolat B, Paglin S, Palmer GE, Papassideri I, Parkes M, Perlmutter DH, Perry G, Piacentini M, Pinkas-Kramarski R, Prescott M, Proikas-Cezanne T, Raben N, Rami A, Reggiori F, Rohrer B, Rubinsztein DC, Ryan KM, Sadoshima J, Sakagami H, Sakai Y, Sandri M, Sasakawa C, Sass M, Schneider C, Seglen PO, Seleverstov O, Settleman J, Shacka JJ, Shapiro IM, Sibirny A, Silva-Zacarin EC, Simon HU, Simone C, Simonsen A, Smith MA, Spanel-Borowski K, Srinivas V, Steeves M, Stenmark H, Stromhaug PE, Subauste CS, Sugimoto S, Sulzer D, Suzuki T, Swanson MS, Tabas I, Takeshita F, Talbot NJ, Tallóczy Z, Tanaka K, Tanaka K, Tanida I, Taylor GS, Taylor JP, Terman A, Tettamanti G, Thompson CB, Thumm M, Tolkovsky AM, Tooze SA, Truant R, Tumanovska LV, Uchiyama Y, Ueno T, Uzcátegui NL, van der Klei I, Vaquero EC, Vellai T, Vogel MW, Wang HG, Webster P, Wiley JW, Xi Z, Xiao G, Yahalom J, Yang JM, Yap G, Yin XM, Yoshimori T, Yu L, Yue Z, Yuzaki M, Zabirnyk O, Zheng X, Zhu X, Deter RL.
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA. [email protected]
Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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The importance to read instruction manual
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Basic experimental techniques (1-10 or more) (enough or not enough?)
The principle behind the technique and its limitation --- ( 君子務本 , 本立道生 ) determination of protein concentration measurement of cell viability, mobility, ROS…. The “feeling” about a series of steps in performing one experiment The ability to foresee results --- Expectation
The “control” group --- how many controls should be done in one experiment?
Authority (it must be….. 想當然耳…… .)
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Example: measuring kinase activity in cells
IP
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The “feeling” about a series of steps in performing one experiment
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Kinase assay in immunoprecipitate (IP) Cells
*homogenization (10-cm dish/0.5 ml lysis buffer)*centrifugation (12000~15000 rpm, 15 min, 4oC)
Supernatants*protein concentration determination*1 mg protein/0.5 ml extracts
*add Ab against specific kinase (5 g)*incubation (1 h, 4oC) *add protein A/G-S4B (50% v/v, 25 l, shaking)
*centrifugation (6000 rpm, 1min, 4oC)*wash/cfg 3 times in Buffer B
Immunoprecipitates*suspended in 20 l Buffer A*substrate (5-10 g), [-32P]ATP.Mg2+ (0.2-20 mM)*shaking for 10-30 min at RT*adding SDS-sample buffer
SDS-PAGE
Autoradiography
Lysis buffer-----10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride,0.5 mg/ml aprotininBuffer A --- 20 mM Tris-HCl at pH 7.0, 0.5 mM dithiothreitolBuffer B --- 0.5 M NaCl in buffer A
cfg
Kinase assay in immunoprecipitate (IP) Cells
*homogenization (10-cm dish/0.5 ml lysis buffer)*centrifugation (12000~15000 rpm, 15 min, 4oC)
Supernatants*protein concentration determination*1 mg protein/0.5 ml extracts
*add Ab against specific kinase (5 g)*incubation (1 h, 4oC) *add protein A/G-S4B (50% v/v, 25 l, shaking)
*centrifugation (6000 rpm, 1min, 4oC)*wash/cfg 3 times in Buffer B
Immunoprecipitates*suspended in 20 l Buffer A*substrate (5-10 g), [-32P]ATP.Mg2+ (0.2-20 mM)*shaking for 10-30 min at RT*adding SDS-sample buffer
SDS-PAGE
Autoradiography
Lysis buffer-----10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride,0.5 mg/ml aprotininBuffer A --- 20 mM Tris-HCl at pH 7.0, 0.5 mM dithiothreitolBuffer B --- 0.5 M NaCl in buffer A
cfg
Basic experimental techniques (1-10 or more) (enough or not enough?)
The principle behind the technique and its limitation --- ( 君子務本 , 本立道生 ) determination of protein concentration measurement of cell viability, mobility, ROS…. The “feeling” about a series of steps in performing one experiment The ability to foresee results --- Prediction/Expectation
The “control” group --- how many controls should be done in one experiment?
Authority (it must be….. 想當然耳…… .)
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The ability to foresee results --- Prediction/Expectation
Exp. 1
Yes No ?Re-design
Exp. 2
Yes No ?Re-design
Exp. 3
Yes No ?Re-design
Exp. 4, 5, 6 and so on…
Basic experimental techniques (1-10 or more) (enough or not enough?)
The principle behind the technique and its limitation --- ( 君子務本 , 本立道生 ) determination of protein concentration measurement of cell viability, mobility, ROS…. The “feeling” about a series of steps in performing one experiment The ability to foresee results --- Expectation
The “control” group --- how many controls should be done in one experiment?
Authority (it must be….. 想當然耳…… .)
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The “control” group --- how many controls should be done in one experiment?
Changing only one parameter in a single exp. at a time
MYO18A PIX
GIT
1
PAK2
Example: identification of novel interacting proteins of a target in cells
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Basic experimental techniques (1-10 or more) (enough or not enough?)
The principle behind the technique and its limitation --- ( 君子務本 , 本立道生 ) determination of protein concentration measurement of cell viability, mobility, ROS…. The “feeling” about a series of steps in performing one experiment The ability to foresee results --- Expectation
The “control” group --- how many controls should be done in one experiment?
Authority (it must be….. 想當然耳…… .)
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【來源：中國華文教育網 】
——宋•張載《經學理窟•義理篇 》
[ 解讀 ]
能在別人不會懷疑的地方提出疑問，這才會有長進。 愛因斯坦曾說過，提出問題比解決問題更為重要。 不發現問題就談不上解決問題，也就談不上有什麼進步。 當然，僅僅能提出一些人人都能提出的問題也是沒有什麼長進的。
“學則須疑” ,“於不疑處有疑方是進矣”
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【來源：中國華文教育網 】
——宋•張載《經學理窟•義理篇 》
[ 解讀 ]
能在別人不會懷疑的地方提出疑問，這才會有長進。 愛因斯坦曾說過，提出問題比解決問題更為重要。 不發現問題就談不上解決問題，也就談不上有什麼進步。 當然，僅僅能提出一些人人都能提出的問題也是沒有什麼長進的。
“學則須疑” ,“於不疑處有疑方是進矣”
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Should be very helpful in:
1. Formation of novel idea in a research field --- ribozyme2. Trouble-shooting for experimental details ---3. ……
What can we learn from our supervisor ???
Basic experimental techniques (1-10 or more) (enough or not enough?) (instruction manual)
The topic(s) and the problem(s) in which that the supervisor is interested (how important and what is the interesting point) (‘good’ or ‘good enough’ science, the wider range of impact)
The way about the formation of the question (hypothesis) that the supervisor wants to address (to solve a important question remained unanswered or just for fun……)
The thinking logic that the supervisor has applied to solve the problem (read a lot of papers and then point out 1-10 methods or just by intuition……)
The way (experimental techniques and attitude) to solve the problem (right methods? Insist or compromise when encounter difficulty? how detail he(she) wants to ask into? how does he(she) explain the data? the attitude about the “truth”)
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The Scientist 2000, 14(14):31OPINIONDistinguishing 'Good' Science from 'Good Enough' Scienceby Gregory J Feist
By ‘good’ I mean a scientist who does work that has more of an impact than that the work of most of his or her peers ---- GJ Feist
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What can we learn from our supervisor ???
Basic experimental techniques (1-10 or more) (enough or not enough?) (instruction manual)
The topic(s) and the problem(s) that the supervisor is interested in (how important and what is the interesting point) (‘good’ or ‘good enough’ science, the wider-range of impact)
The way about the formation of the question (hypothesis) that the supervisor wants to address (to solve a important question remained unanswered or just for fun……)
The thinking logic that the supervisor has applied to solve the problem (read a lot of papers and then point out 1-10 methods or just by intuition……)
The way (experimental techniques and attitude) to solve the problem (right methods? Insist or compromise when encounter difficulty? how detail he(she) wants to ask into? how does he(she) explain the data? the attitude about the “truth”)
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What can we learn from our supervisor ???
Basic experimental techniques (1-10 or more) (enough or not enough?) (instruction manual)
The topic(s) and the problem(s) that the supervisor is interested in (how important and what is the interesting point) (‘good’ or ‘good enough’ science, the wider-range of impact)
The way about the formation of the question (hypothesis) that the supervisor wants to address (to solve a important question remained unanswered or just for fun……)
The thinking logic that the supervisor has applied to solve the problem (read a lot of papers and then point out 1-10 methods or just by intuition……)
The way (experimental techniques and attitude) to solve the problem (right methods? Insist or compromise when encounter difficulty? how detail he (she) wants to ask into? how does he (she) explain the data? the attitude about the “truth”)
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What can we learn from our supervisor ???
Basic experimental techniques (1-10 or more) (enough or not enough?) (instruction manual)
The topic(s) and the problem(s) that the supervisor is interested in (how important and what is the interesting point) (‘good’ or ‘good enough’ science, the wider-range of impact)
The way about the formation of the question (hypothesis) that the supervisor wants to address (to solve a important question remained unanswered or just for fun……)
The thinking logic that the supervisor has applied to solve the problem (read a lot of papers and then point out 1-10 methods or just by intuition……)
The way (experimental techniques and attitude) to solve the problem (right methods? Insist or compromise when encounter difficulty? how detail he (she) wants to ask into? how does he (she) explain the data? the attitude about the “truth”)
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Other sources for learning scientific methods??
Other experienced investigators in your/other lab
(which one is the best for you to learn?)
(the one who succeeds or fails in doing each experiment)
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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Can we learn by ourselves ???
Do you always keep in mind and think about your problems ?
Do you really recognize the fundamentals of your problems ? How to analyze ?
Do you know all of the methods that can be potentially applied to solve your problems ? How to know ?
Have you ever created any new idea in doing experiments (generate a new question/hypothesis or think about a new method) ?
Have you ever tried to establish a new method in you lab ? When and how ?
Free thinking to link two or more things together
How you describe your experiments in terms of novelty, importance and feasibility ?
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The End
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