i need help please ??This week we are going to be reading some fictional short stories for our Monsters Unit. The story “Eight Bites” addresses one such danger, the toxic messages women internalize about body image (Links to an external site.), which contribute to, if not engender, the various pathological conditions described in the Diagnostic and Statistical Manual (Links to an external site.) of Psychological Disorders, Fifth Edition. While it is difficult to say whether the protagonist of “Eight Bites” has Binge Eating Disorder (Links to an external site.) or Body Dysmorphic Disorder (Links to an external site.), her relationship to food and eating is unhealthy to such a degree that she undergoes bariatric surgery, with no indication whatsoever that this procedure is medically necessary.Carefully read through “Eight Bites” by Carmen Maria Machado. Read it slowly! Read it twice! Use the questions above to help you annotate. This reading I am providing as a link because there are videos embedded in the article.ccmachado.docxMinimize File PreviewFill out an annotation worksheet for the reading.Blank Annotation Worksheet here. (DOC)Blank Annotation Worksheet here. (PDF)(You can download it, type in your responses, and save your file or you can print it out, write your answers on the worksheet, and post a scan of the document.)Submission:To submit, please attach your saved files as either a Word, PDF, JPG or PNG file below and click Submit. You can submit twice for each annotation worksheet.
CHARACTERIZATIONOF Α-LACTALBUMINRECORDWeek 7GoalsDiscuss Exp 4Watch “Exp 4b sephadexchromatography”Analyze the elution profileWeeks/Dates Experiment # Report DueDatesWeek1 (8/31-9/04) Lab check in, lab safety training, Experimental Design, Review ofconcentration terms, Accuracy and Precision & Pipetting techniqueWeek2 (9/7-9/11) Exp 1: Protein determination; Lowry & Bradford assaysWeek3 (9/14-9/18) Exp 2: Amino acid titration & Making a bufferExp1 FirstDraftExp2 (in-classworksheet)Week4 (9/21-9/25) Exp 3a: Ion exchange chromatography Exp 1Week5 (9/28-10/2) Exp 3b: Paper chromatographyWeek6 (10/5-10/9) Exp 4a: Purification of α-lactalbumin from milk: Milk Whey production& chromatography column preparationExp3 FirstDraftWeek7 (10/12-10/16) Exp 4b: Size exclusion chromatography Exp 3Week8 (10/19-10/23 Exp 4c: SDS-PageWeek9 (10/26-10/30) Exp 4d: Western blottingWeek10 (11/2-11/6) Exp 5a: Enzyme Kinetics: Rate of Invertase Reaction Exp4 FirstDraftWeek11 (11/9-11/13) Exp 5b: Enzyme kinetics: Determination of kinetic parameters andEffect of enzyme inhibitor Exp 4Week12 (11/16-11/20) NO LAB: Use this time to work with your partner on Exp 5 report Exp5 FirstDraftWeek13 (11/23-11/27) NO LAB: Thanksgiving holidayWeek14 (11/30-12/4) LAB FINAL EXAM Exp 5Week15 (12/7-12/11) NO LABDue today: Exp 3 ReportCheck the file afteryou upload, beforethe assignment duedate!!!3 weeks later: Exp 4 First DraftExp 4 Purpose: Purify andcharacterize a-lactalbumin◦ Week 1: isolate whey from non-fat milk◦ Week 2: size exclusion chromatography◦ separate whey proteins based on size◦ Week 3: SDS PAGE◦ Separate proteins based on size◦ Week 4: Western Blotting◦ Detect the a-lactalbumin using the specific antibodypurify a-lactalbuminAnalysis (purity, yield)Size Exclusion (Gel Filtration)Chromatography◦ Stationary phase: beads of ahydrated, sponge-like materialwith pores◦ Small molecules enter thepores and move slowly downthe column. Elute LATER.◦ Large molecules do not getcaught in the pores, andhence move quickly down thecolumn. Elute FIRST.S.G. Prapulla, N.G. Karanth (2014)https://www.youtube.com/watch?v=oV5VB5kO3tQStationary Phase(Sephadex)◦Beads that consist of cross-linked dextran (a complexbranched glucan derived from the condensation ofglucose)◦Sephadex G-50 is able to separate proteins with thesize range of 1.5 k – 30 kDa.α1α6α3α1Packing the column with Sephadex◦ For M&M, discuss the column packing and running protocol together underthe size exclusion chromatography. Do not separate by “weeks”. (Packingthe column does not take a “week”.)◦ You are packing the column with beads today. What are the factors that canlater affect the protein separation during the elution?A. To speed up the packing, pressure was applied to the column to increase the flowrate.B. The buffer ran out during the packing. The beads became dry.C. Beads were poured little by little until the desired volume was reached. They were notadded all at once.D. Excess beads had to be removed from the top because too much beads wereadded.E. The packed column was stored at 4 °C for a month, then was used for the actualseparation experiment.Factors Affecting Size Exclusion (GelFiltration) Chromatography Elution•Increased column length enhances resolution•Slower flow rate, better resolution•Sample diffusion during the loading will cause thedownstream band broadening.•Different beads have different pore sizes, anddifferent separation ranges (Sephadex G-25, 50,100, etc.)Size exclusion chromatography◦ Separate the whey proteins based on the sizeWhey proteins w/w Molecular weight (kDa)β-lactoglobulin 65 % 18.4α-lactalbumin 25 % 14.2BSA (bovine serum albumin) ~8 % 66.5immunoglobulins ~2 % 150Monitor protein elution with A280◦ A=klc◦ AA with aromatic rings (Trp, Tyr) has λmax at 280 nm◦ Phe – relatively insignificant absorption◦ If the number of residues for Trp and Tyr is known, rough estimate ofthe protein concentration may be made.◦ Fast and convenient. No additional reagent needed.Column Elution profileElutant (mL)A280Whey proteins w/w MW (kDa)β-lactoglobulin 65 % 18.4α-lactalbumin 25 % 14.2BSA ~8 % 66.5immunoglobulins ~2 % 150Column Elution profileElutant (mL)A280Whey proteins w/w MW (kDa)β-lactoglobulin 65 % 18.4α-lactalbumin 25 % 14.2BSA ~8 % 66.5immunoglobulins ~2 % 150Assessing the proteins in elution fractions◦ Purity◦ High purity – only the protein of interest is present◦ Low purity – contains more than 1 protein◦ Overlapping protein peaks indicate the mixture of proteins◦ Yield (amount)◦ A280 is proportional to protein concentration◦ apex of the elution peak has the highest protein concentrationWhich fraction has the most pure a-lactalbumin? B-lactoglobulin?Which fraction has the highest amount of a-lactalbumin? B-lactoglobulin?Column Elution profileElutant (mL)A280Whey proteins w/w MW (kDa)β-lactoglobulin 65 % 18.4α-lactalbumin 25 % 14.2BSA ~8 % 66.5immunoglobulins ~2 % 150Column Elution profileElutant (mL)A280Whey proteins w/w MW (kDa)β-lactoglobulin 65 % 18.4α-lactalbumin 25 % 14.2BSA ~8 % 66.5immunoglobulins ~2 % 150
Plot an elution profile• A280 vs elutant (mL)
Identify the elution peaks for the whey proteins•Larger proteins elute first, smaller proteins elute last.•A280 is proportional to the protein concentration
Assess the efficacy of protein separation• Which fraction has the highest concentration of a-lactalbuminor B-lactoglobulin?• Are a-lactalbumin or B-lactoglobulin fractions pure?• Think about what could have been done to improve this.Exp 4 Purpose: Purify andcharacterize a-lactalbumin◦ Week 1: isolate whey from non-fat milk◦ Week 2: size exclusion chromatography◦ separate whey proteins based on size◦ Week 3: SDS PAGE◦ Separate proteins based on size◦ Week 4: Western Blotting◦ Detect the a-lactalbumin using the specific antibodypurify a-lactalbuminAnalysis (purity, yield)
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