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- SDS-PAGE reagents that play a role in formation of the gel only are(Select all that applies) Beta-Mercaptoethanol Bromophenol blue APS Heat Sodium Dodecyl Sulfate Bis-acrylamide Acrylamide TEMED21. R Rhus toxicodendron extract 10 mg/ 100 mL Sterile water for injection q.s. Sig: as directed How many milliliters of a 100 mg/mL concentrate of Rhus toxicodendron extract should be used in preparing the prescription?Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)1. What are the functions of the sample buffer and sample reducing agent? Why do the samples need to be heated before you load them on the gel?2. What determines the current in your gel? What could cause the current in your gel unit to be lower than expected? Is there anything that could cause it to be higher than expected?3. Why do you need to wash the gel before staining it? Why use warm water?
- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 1. What determines the current in your gel? What could cause the current in your gel unit to be lower than expected? Is there anything that could cause it to be higher than expected? 2. Why do you need to wash the gel before staining it? Why use warm water?4.outline the processes used in the preparation of agarose gel of 1.5 concentration 5.outline the procedures /steps in gel electrophoresis 6.how do we prepare x1(concentration) TAE buffer from 50x TAE bufferExample of a Protein Purification Scheme: Purification of the Enzyme Xanthine Dehydrogenase from a Fungus Volume Total Total Specific Percent Fraction (mL) Protein (mg) Activity Activity Recovery 1. Crude extract 2. Salt precipitate 3. Ion-exchange chromatography |4. Molecular-sieve chromatography 5. Immunoaffinity chromatography 3,800 22,800 2,460 0.108 100 165 2,800 1,190 0.425 48 65 100 720 7.2 29 40 14.5 23 1.8 275 152.108 11 Calculate the specific activity of step#4. Note that percent recovery=% Yield.
- Prepare the following LB media with antibiotic added. A 100ml of LB media with 25 micro/ml of Amp and 100micro/ml of Kan final concentration. A 100ml of LB is provided and amp of Kan stock at 100 mg/ml and 50 mg/ml are also provided. Find how much each antibiotic stock solution is needed to add 100ml of LB to reach desired antibiotic concentration.Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 1. Why do you destain the gel? 2. What is a gradient gel? Why are we using them? 3. Compare and contrast SDS-PAGE and native PAGE. Why would we want to do each of these techniques?1) You have been asked to make up four 1.5% agarose gels at 30 ml each. Ethidium Bromide is to be added at 0.5 ug/ml and you have a bottle at 10ug/ml. Write down step-by-step protocol of how to proceed. 2. How would you make up one liter of 1X TAE buffer using a 25X stock?
- In SDS PAGE the resolving gel : is 4%(w/v) acrylamide,PH )6.8 is 6-20% (w/v) acrylamide is 4%(w/v) acrylamidea. State the importance of using following reagents in SDS-PAGE. 1. Acrylamide 2. Bisacrylamide 3. Tetramethylethylelediamine 4. Glycerol 5. Ammonium persulfate b. Briefly describe the importance of two dimensional electrophoresis in protein separation?ANSWER THE FOLLOWING QUESTIONS REGARDING GEL ELECTROPHORESIS 1.why is it not advisable to move/touch the agarose gel in the process of hardening 2.what is the use or function of the TAE buffer that is poured or found in the gel box 3.how can you tell if agel is running 4.outline the process use in the preparation of agarose gel of 1.5 concentration 5.outline the processes/steps used in gel electrophoresis 6.name twoexamples of the dye used in gel electrophoresis 7.how do we prepare x1(concentration) TAE BUFFER form 50x TAE buffer