Microsoft PowerPoint - Practical report Information.pptx Practical report • Due by 5pm Tuesday 10 September 2019 (Week 9) via our Cloud site • 20% final mark • 2500 word limit, excluding references,...

The report aim is isolation of mitochondria from liver and bca protein. I will send in the prac manual later.


Microsoft PowerPoint - Practical report Information.pptx Practical report • Due by 5pm Tuesday 10 September 2019 (Week 9) via our Cloud site • 20% final mark • 2500 word limit, excluding references, headings and figure legends • Guidelines and rubric in Assessment folder on SLE206 Cloud site • Questions from datasheets / manual must be answered within the report • Can be incorporated into the report or in a separate section Practical report – general style • High quality • Professional • Scientific • Succinct • Content needs to be comparable to a research publication / manuscript Layout • Title • Concise but informative • Aims • What were you trying to achieve, specifically? • Introduction • Background and rationale, hypothesis • Keep it relevant • Refer to related research • Note: Aims can be included as a separate section OR at the end of the introduction, as long as it’s clear Materials and Methods • Description of the procedure or protocols you used • Description of the methods you used to analyse your results • Describe what you did to the point where it could be replicated • Do not list reagents or equipment in dot point form, write it in paragraph form but be concise • Refer to published research articles for examples on how to achieve this Results • Written description of the results you obtained referring to the data • Present data in the form of tables, graphs and/or figures • Ensure all tables and figures are referred to in the text • Be careful not to repeat your methods • Do not discuss your results Discussion • Discuss the meaning of your results • Discuss how this is significant or important • Refer to findings of others • Highlight any limitations, if any, of your experiments or why your results weren’t what you expected • Mention any future perspectives • Ensure you don’t present any new results and be careful not to repeat your results in any great detail • May be a good section to answer some of the questions Tables, Figures and Figure Legends • Present your data in the form of figures, tables or graphs • Make sure each figure/table has a figure/table number and this is referred to throughout the report • Make sure figures/tables are numbered in order • Make sure a figure/table legend is present and clear • A figure legend should stand alone – a reader should be able to get all the information about the figure from the legend without referring to the main text. So it should have a title, a brief description of how the results were obtained and how the data was analysed. Do not discuss the results in the figure legend, just state what the figure shows. Referencing • Use a standard style of your choice • Ensure the referencing style is CONSISTENT throughout the report • Ensure all references in the text are included in the reference list and vice versa • The use of the referencing program EndNote is encouraged – talk to the library about getting help with this Microsoft PowerPoint - Mitochondrial Isolation Practical 2 - Demo Slides.pptx Practical 2 ISOLATION OF MITOCHONDRIA FROM LIVER  AND BCA PROTEIN ASSAY  PART 1 SLE206 T2 2019 Teaching Objectives i) To familiarise students with the methods used to isolate  subcellular fractions from cells. ii) To demonstrate how enzymatic assays can be used for  identification of the components of the subcellular fractions. iii) To gain experience using laboratory skills that are desired by  potential employers. Mitochondria • Organelle with double  membrane • Bulk of the cells ATP  production as part of  cellular respiration • Involved with many other  biochemical functions of  the cell http://www.news‐medical.net/life‐sciences/What‐are‐Mitochondria.aspx Mitochondria • The aim of this experiment is to separate the components of the liver  cells based on size using differential centrifugation • We will then determine which components contain succinate  dehydrogenase and thus identify which fraction(s) contain  mitochondria • Succinate dehydrogenase: • Located within the inner membrane of the mitochondria • Participates in both the electron transport chain and citric acid cycle (cellular  respiration)  • Commonly used as a marker for mitochondria Differential Centrifugation BCA Assay – Standard Curve • Standard curve is plotted using assayed protein samples of a known  concentration against their absorbance and generating a line of best fit • This generated a ‘Standard Curve’ that can be used to estimate the concentration  of protein in a sample with an unknown concentration based on its absorbance  when assayed Protein standards – known concentration of  BSA – prepared by serial dilutions Standard Curve – absorbance of standards  plotted against concentration 0 2000 Tube Volume of PBS (µL) Volume of BSA solution (µL) Final BSA concentration (µg/mL) A 300 0 0 B 0 300 of stock (2 mg/ml) 2000 C 125 375 of stock (2 mg/ml) 1500 D 325 325 of stock (2 mg/ml) 1000 E 175 175 tube C 750 F 325 325 tube D  500 G 325 325 tube F 250 H 325 325 tube G 125 I 400 100 tube H 25 Table 1 Bovine Serum Albumin (BSA) standards How to prepare protein standards Note The unused wells will be used by the next class so please start with first available row on the plate. Also be careful not to contaminate other wells. Prac 3: To check mitochondria purity • There are quite a few  enzymes that can be  used to test  mitochondrial quality.  • Any part of the TCA  cycle can be used as an  indicator of  mitochondrial activity. Practical Overview – Part 1 & 2 Whole cell lysate  prepared from toad liver  (technical staff) Subcellular fractions  isolated via differential  centrifugation Bradford assay:  preparation of standard  curve based on BSA  standards Bradford assay: protein  concentration of each  fraction determined  from standard curve Calculate 50 ug total  protein from each  fraction Analyse each fraction for the  presence of succinate  dehydrogenase (succinate  dehydrogenase assay) Microsoft PowerPoint - Succinate Dehydrogenase Assay Practical 3 - Demo Slides.pptx Practical 3 ANALYSIS OF SUBCELLULAR FRACTIONS FOR  INTACT MITOCHONDRIA (SUCCINATE  DEHYDROGENASE ASSAY) PART 2 SLE206 T2 2019 Mitochondria • Organelle with double  membrane • Bulk of the cells ATP  production as part of  cellular respiration • Involved with many other  biochemical functions of  the cell http://www.news‐medical.net/life‐sciences/What‐are‐Mitochondria.aspx Tube Volume of PBS (µL) Volume of BSA solution (µL) Final BSA concentration (µg/mL) A 300 0 0 B 0 300 of stock (2 mg/ml) 2000 C 125 375 of stock (2 mg/ml) 1500 D 325 325 of stock (2 mg/ml) 1000 E 175 175 tube C 750 F 325 325 tube D  500 G 325 325 tube F 250 H 325 325 tube G 125 I 400 100 tube H 25 Table 1 Bovine Serum Albumin (BSA) standards How to prepare protein standards Note The unused wells will be used by the next class so please start with first available row on the plate. Also be careful not to contaminate other wells. How to prepare your standard curve Absorbance (595nm) Conc. Of BSA  (µg/ml) 0 0 2.174 2000 1.79 1500 1.309 1000 1.066 750 0.643 500 0.384 250 0.201 125 0.048 25 0 500 1000 1500 2000 2500 0 0.5 1 1.5 2 2.5 Co nc . o f B SA  (µ g/ m l) Absorbance (595nm) Conc. Of BSA (µg/ml standard curve plot) To check mito purity…….. There are  quite a few  enzymes that  can be used  to test  mitochondrial  quality. Any  part of the  TCA cycle can  be used as an  indicator of  mito activity Mitochondria • Succinate dehydrogenase: • Located within the inner membrane of the mitochondria • Participates in both the electron transport chain and citric acid cycle (cellular  respiration)  • Commonly used as a marker for mitochondria • The aim of this experiment is to separate the components of the liver  cells based on size using differential centrifugation • We will then determine which components contain succinate  dehydrogenase and thus identify which fraction(s) contain  mitochondria Succinate Dehydrogenase Assay • In aerobic cells, Succinate Dehydrogenase catalysed the oxidation of  succinate to fumarate: • This reaction occurs in the innermitochondrial membrane and is part of  the citric acid cycle during cellular respiration Succinate Dehydrogenase Assay • This reaction involved the transfer of hydrogen from succinate to FAD  giving rise to FADH2 • We can assay the activity of Succinate Dehydrogenase by using an artificial  electron acceptor, p‐iodonitrotetrazolium (INT) in place of FAD Succinate Dehydrogenase Assay • This reaction involved the transfer of hydrogen from succinate to FAD  giving rise to FADH2 • We can assay the activity of Succinate Dehydrogenase by using an artificial  electron acceptor, p‐iodonitrotetrazolium (INT) in place of FAD • This reaction can be observed by the naked eye as the solution turns from  colourless to rusty red/pink Succinate Dehydrogenase Assay Therefore, a colour change is  an indication of the presence  of mitochondria This can be measured  spectrophotometrically  Clear = No Mitochondria Major Colour Change = High Levels of  Mitochondria Minor Colour Change  = Low Levels of  Mitochondria Beer‐Lambert law Practical Overview – Part 1 & 2 Whole cell lysate  prepared from toad liver  (technical staff) Subcellular fractions  isolated via differential  centrifugation Bradford assay:  preparation of standard  curve based on BSA  standards Bradford assay: protein  concentration of each  fraction determined  from standard curve Calculate 50 ug total  protein from each  fraction Analyse each fraction for the  presence of succinate  dehydrogenase (succinate  dehydrogenase assay)