Biol 3360 Spring 2008 - Chicken Muscle AMP Aminohydrolase

Tue, 06/04/2013 - 13:33 -- sewm02

Biol 3360 Spring 2008 - Chicken Muscle AMP Aminohydrolase

 

This Wiki will be used by the participants of the Biochemistry Laboratory Biol 3360 during Spring 2008 to collaborate in the pre-laboratory work of the "wet" portion of the course.

It is recommended that the students add this page to their watchlist to make sure they are informed when information is added to the page. The instructor will post comments in the corresponding discussion tabs.

Task Distribution

general instructor comment: How are you going to do the steps in which you have to adjust the pH with Tris?

Extraction Buffer - Jordan and Greg

The following calculations are used to determine the mass of the chemicals that will be needed to prepare the extraction buffer at a volume of 1000mL. The chemicals will be weighed and added to a 1000mL volumetric flask and diluted to the graduated line with distilled water.

 

Actual Procedure: 9.3668g of Fisher Scientific K2HPO4, 4.230g of J.T. Baker Chemical Co. KH2PO4, and 13.3524g of Fisher Scientific KCl were placed in a 1000 mL beaker. Roughly 600 mL of distilled water was added to the beaker. The solution was stirred on a magentic stir plate. The starting pH of the solution was 6.976 and the desired pH level was 6.5. In order to lower the pH, 3M HCl was added drop wise until the pH was 6.507. The solution was then transfered to a 1000 mL volumetric flask and diluted to the line with distilled water. The solution was placed in a jar for storage.

Pyrophosphate Buffer - Amanda, Megan, Tiffany

Going to need 200 ml for each group. We are going to go ahead and make 500 ml.

Actual procedure: Weighed out 16.777 +/- 0.003 g of KCl, 4.1298 +/- 0.0003 g of potassium pyrophosphate, and 2.4430 +/- 0.003 g of MES and placed into a 250 ml beaker. Added about 250 ml of distilled water and stirred using a magnetic stirrer. Measured the pH using a pH probe. The pH was 7.1. Then 35 ul of 2-mercaptoethanol was added using a micropipette. The solution was transferred to a 500 ml volumetric flask and diluted to the fill line with distilled water. It was placed into a jar for storage.

Assay Solution - John Pat, Aaronh

KCL is added to attain a pH of 6.5

Actual Procedure: In a 300-ml beaker, 0.4574 g of AMP was added, along with 2.80g of solid KCl and 2.44g of MES. Approximately 200 ml of distilled water was added. Solid Tris was added to attain a pH of approximately 6.5, at which time the solution was transferred to to a 250-ml volumetric flask. Distilled water was added to the flask until a level 250 ml was attained. The assay solution was then transferred to a labeled plastic bottle and stored in the Organic Chemistry freezer.

MES-Tris Wash Buffer - Whitney, Leslie

We're going to need approximately 700 mL for both groups, so we're going to go ahead and make 1 L.

Calculations:

(0.45 mol / L)(74.54 g / L)(1 L) = 33.543 g KCl

(25 mmol / L)(1 mol / 1000 mmol)(213.25 g / mol) = 5.331 g MES

(1 mmol/L)(1 mol/1000 mmol)(74.14 g/mol)(1 mL/1.114 g)(1000 uL/1 mL) = 66.6 uL 2-mercaptoethanol

Acutal Procedure: 35.558 g of KCl and 4.882 g of MES were combined in a 1000 mL beaker. Approximately 800 mL of distilled water was added to the beaker. The beaker was placed on a stir plate and a stir bar was added. The solution was continuously stirred while solid tris was slowly added to adjust the pH to 6.5. The solution was transferred to a 1000 mL volumetric flask. Distilled water was added to the line. Under the hood, 70 uL of 2-mercaptoethanol were added to the solution. The solution was labeled and stored for the next lab.

Phosphocellulose - Alyssa, Erin

Pre-cycling: 680 g of cellulose phosphate, 1 L of 0.5 M NaOH, 1 L of 0.5 M HCl

For NaOH: 500 mL of 1.0 M NaOH, 500 mL of dH2O

For HCl: 400 mL of 0.5 M HCl, 300 mL of 1.0 M HCl, 300 mL of dH2O

 

For Batch Separations: 1 L total, 1.35 M KCl and 75 mM MES

For KCl: (1.35 mol/L)(74.551 g/mol) = 100.644 g/L

For MES: (75 mmol/L) (1 mol/ 1000mM) (195.24 g/mol) = 14.64 g/L

Actual Procedure: Made 1 L of NaOH and Hcl solutions using the recipes above in volumetric flasks. For the batch separation buffer, 100.64 g of KCl and 14.64 g of MES were measured and dissolved in 900 mL of dH2O in a 1000 mL beaker using a magnetic stir bar and plate. The pH was adjusted to 6.5 by adding tris while monitoring with a pH probe. Once the pH reached 6.5, the solution was poured into a volumetric flask and dH2O was added until the solution reached the fill line. All the solutions were transfered to jars for storage.

Week 2 of Lab (01-31-08)

Group 1 (Megan, Amanda, Tiffany, Jordan, Greg, Alyssa)

Chicken Soup

Megan, Amanda, and Alyssa will be making the "chicken soup." We are going to take 100 g of fresh chicken breast, partially thawed, and cut into small pieces (about 1 cm cubes). Then place the chicken into a blender with 400 ml of cold extraction buffer. It will be blended at high speed with five 15 second bursts. The homogenate is then centrifuged at 6000 x g for 15 minutes. The supernatant is decanted through a layer of glass wool and the rest discarded. The volume of the supernatant is recorded. About 0.5 ml is removed for the enzyme assay and protein determination, and 20 ml of washed phosphocellulose is added to the homogenate. The mixture is stirred for 1 hour.

Phosphocellulose

Jordan, Greg, and Tiffany will be making the phosphocellulose.

Stir the weighed cellulose phosphate into 25mL of .5M NaOH and let sit for five minutes. Filter off the supernatant and wash the medium with distilled water in a separatory funnel. The mixture in the separatory funnel should separate out into two different layers, assuming the H2O is on the bottom and the cellulose phosphate is on the top, drain out water layer into a beaker. Using litmus papers test the pH making sure the pH is below 11. If this pH is not below 11, continue the process until the desired pH is reached.

Then mix the cellulose phosphate solution with 25mL of .5M HCl and let is sit for five minutes. Filter off the supernatant and wash the medium with distilled water into a separatory funnel. The mixture should separate out the same as before, drain the bottom layer into a beaker. Test to see if the pH is above 3 using litmus paper. If it isn’t continue desired process until desired process is reached.

If filtering yields poor results we will try to decant the solution.

Enzyme Assay

At various time intervals, aliquots of 0.5 ml are removed from the phosphocellulose and homogenate solution and the phosphocellulose is removed by centrifuge. The supernatant is assayed to determine loss of activity relative to the crude extract. About 80% of the enzyme will be bound within an hour. The phosphocellulose is allowed to settle for 15 minutes and the supernatant is decated and an aliquot is saved for assay. The phosphocellulose slurry is vacuum filtered on a 500 ml Buchner funnel and is successively washed with 3 portions of 100 ml of extractioin buffer, and two 100 ml portions of MES-Tris wash buffer. The phosphocellulose should not be allowed to dry completely throughout the procedure. The volume of the washes will be recorded and the fraction checked for enzyme activity. A slurry of the phosphocellulose will be made by the addition of some of the MES-Tris wash buffer. The slurry will be stored until the next week (I think). If the slurry is not stored until next week, the slurry will be poured into a column (2x10Cm). The column will be washed with 200 mL of MES-Tris wash buffer at a rate of 1 mL/min. The elute is collected in a beaker, and then possibly stored.===FEEL FREE TO MAKE CHANGES GROUP MEMBERS!!!!===

 

Actual Procedures

'Extraction Buffer Procedure

In order to prepare the chicken AMP extraction buffer, 9.3668g of Fisher Scientific K2HPO4, 4.4230g of J.T. Baker Chemical Co. H2KPO4, and 13.3524g of Fisher Scientific KCl were added to a 1000mL volumetric flask. Roughly 500mL of distilled water was added to the flask, which was then stirred on a stir plate using a stir rod. A Vernier pH Sensor was attached to a Texas Instruments TI-84 calculator in order to monitor the pH value of the buffer. Under the Application menu, DataMate was selected and the pH sensor was calibrated using pre-made solutions of pH’s 1 and 10. Once the voltage stabilized, the pH values of the solutions were entered in the calculator and the Enter button was hit. The starting pH of the buffer was 6.967. 3M HCl was added dropwise until the pH of the buffer was 6.507. Distilled water was added to the flask until the bottom of the meniscus was at the dilution mark. The contents of the flask was transferred to a glass container and placed in the refrigerator until needed.

Affinity Chromatography Procedure and Results

In order to filter the cellulose phosphate, a column was set up. A small portion of glass wool was placed in the bottom of the column, which was held in place on a ring stand. A 250mL beaker was placed below the beaker. The cellulose phosphate was poured into the column followed by Mes-Tris wash buffer. The purpose of the buffer was to wash the phosphate. The cellulose phosphate collected above the glass wool and was a white color. The stop cock was opened and the eluted solution emptied into the beaker. The eluted solution was a yellow color. Samples of the eluted solution were tested until the absorbance was at or below 0.027. An Ocean Optics USB-ISS-UV/VIS USB 4000 was used with LoggerPro software in order to measure the absorbance. The cellulose phosphate was washed three times with Mes-Tris wash buffer before the absorbance was at the desired level. After the cellulose phosphate was washed, 20 test tubes were labeled and placed in an ice bath and pyrophosphate buffer was added to the column. 4mL 0.02 mL samples were obtained in each test tube and the absorbance was recorded.

Chicken Soup:

Fresh chicken breast was cut into small pieces. Exactly 100.4 g of chicken breast pieces were weighed out and placed into the blender. To the blender, 400 mL of cold extraction buffer was added. The mixture was blended using 5 - 15 s bursts. The blended mixture was then poured into 11 – 50 mL centrifuge tubes. All the tubes were filled to the 50 mL line. The tubes were centrifuged at 5100 rpm for 15 minutes at 4°C. The supernatant was decanted through a layer of glass wool, and the volume was recorded to be 116.5 mL. The remaining pellets were discarded.

From the supernatant, 0.5 mL was removed and transferred to a microcentrifuge tube labeled, 0 min. The supernatant was placed into a beaker surrounded by an ice bath and put onto a stir plate. To the beaker, 20 mL of washed phosphocellulose was added. The mixture was allowed to stir for 1 hour. After 30 min., another 0.5 mL was removed and transferred to a microcentrifuge tube labeled 30 min.

Preparation of Dialysis Tubes:

A total of 4 tubes were prepared, 2 for each group. The packages containing the tubes were opened, and the dialysis tubes were removed. While holding onto the collar of the tube, the protective plastic that surrounded the tubes was removed. The 0.1% sodium azide preservative solution was poured out of the protective plastic cover. Then, the cap of the dialysis tube was removed and the 0.1% sodium azide solution in the dialysis tube was removed via pipette. Using a new pipette, the tube was filled with distilled water. The cap was replaced and the tube was put into a container filled with distilled water. The procedure was repeated with the remaining dialysis tubes. Once the four tubes had been prepared, the container with the tubes was placed onto a stir plate, a stir bar was added, and the distilled water with the tubes was stirred for 30 minutes. The tubes were then stored in distilled water in a refrigerator until they were needed.

Absorbance Measurements:

Absorbance of the samples collected during affinity chromatography was measured at 280 nm.

Enzyme Assay:

Based on the absorbance measured, only samples 6-10 were used for the enzyme assay along with samples taken from the chicken soup at 0, 30, and 60 minutes. For the enzyme assay, absorbance was read at 290 nm for 1 minute. Into a cuvette, 2.975 mL of enzyme assay reagent was added. To the cuvette wall, 25 µL of each sample was added. The cuvette was covered with parafilm and inverted to mix.

 

Enzyme Assay:

Based on the absorbance measured, only samples 6-10 were used for the enzyme assay along with samples taken from the chicken soup at 0, 30, and 60 minutes. For the enzyme assay, absorbance was read at 290 nm for 1 minute. Into a cuvette, 2.975 mL of enzyme assay reagent was added. To the cuvette wall, 25 µL of each sample was added. The cuvette was covered with parafilm and inverted to mix.

Image:Enzyme_Assay.jpg

The units/mL of enzyme concentrations was determined by using Excel to put the slopes into the following equation:

Units/mL sample =(change in absorbance per minute at 290 nm)/ (0.12 mM-1cm-1 x mL sample)

Since samples 7 and 8 had the highest enzyme concentration, their volumes were recorded. The volume of 7 was 3.30 mL, and the volume of 8 was 3.09 mL. These samples were put into the prepared dialysis tubes for further purification.

BCA:

There were two samples of which the protein concentration needed to be determined and 6 dilutions were needed to make a standard curve. With a total of 8 tubes, 25 mL of reagent A and 0.5 mL of reagent B were mixed together in a beaker to make the working reagent. The tubes were prepared in the following way:

Image:BCA_1.jpg

Once prepared the samples were placed into a 37°C waterbath for 30 minutes. The absorbance of each solution was read using a reference wave of 562 nm.

Image:BCA_Absorbances.jpg

SDS-Page:

A boiling water bath was set up. Samples were prepared in microtubes. For each sample 41 µL of sample, 5µL if 1xSDS solution, 1 µL of bromophenol blue (BPB) dye, and 3 µL of 2-ME (mercaptoethanol) was added. The tubes were centrifuged to remove excess liquid from the sides. The tubes were put into the boiling water bath for a few minutes while the gels were prepared.

Gels were prepared by wiping the pre-cast polyacrylimide gels off with Chem-wipes. The wells were drawn onto the gel using a marker. These wells were drawn on with the side with tape. The gel was then flipped over and another line was drawn at the top of the tape. This would ensure that the gel did not run to long.

The electrophoresis unit was prepared. The gels were placed with the thick rounded side facing outward. The tape had to be removed before the gels were put into place. The gels were snapped into place. Running buffer was prepared by mixing 50 mL of distilled water with 450 mL of pre-made 10xTris/Glysine/SDS Buffer Solution from BIO RAD Laboratories. (The mixture at 1x contains 25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS.) The running buffer was poured so that between the gels was completely filled. The remaining buffer was added to the outside. Once the buffer was added, the plastic combs were removed from the pre-cast gels.

The gels were loaded with 10 µL of Kalidoscope Size marker and 45 µL of each sample. The gels were loaded as shown in the table below, with Lane 1 appearing on the left. In the wells without size marker or sample, 10 µL of Laemmli Buffer was added. The gels ran at 200 V for 30 minutes packed in ice.

Image:SDS_PAGE.jpg

  • The samples in Lanes 4-6 were taken from another group since the samples collected from the chicken soup were discarded.

After 30 minutes, the gels were removed by breaking the plastic casting. The gels were put into trays and covered with distilled water for 5 minutes. After 5 minutes, the water was poured off and the gels were covered with Bio-Safe Coomassie Stain from BIO RAD Laboratories. The trays with the gels and stain were put onto a rocker plate and remained for one hour. After 1 hour, the dye was poured off and the gels were washed with distilled water. As the gels washed, they were put back onto the rocker plate. A picture of the gels was taken using the BioRad ChemiDoc XRS camera.

Group 2 (Aaron, John, Erin, Leslie, Ian)

Procedure: Chicken Preparation

1. Cut chicken into 1cm cubes. 2. Place 100g into blender with 400ml of extraction buffer. 3. blend with five 15 second bursts 4. Centrifuge the resulting homogenate 6000 x G for 15 minutes 5. Poor resulting supernatant through glass wool, discard pellet 6. Record volume of supernatant. (Remove 5mL for enzyme assay and protein determination).

Procedure: Cellulose phosphate exchanger preparation

1. Weigh out _____ g of cellulose phosphate. 2. Add the cellulose phosphate to 25 volumes of 0.5 M NaOH. 3. Leave for 5 minutes. 4. Filter or decant the supernatant. 5. Wash the medium (with distilled water?) until the pH is 11.0 or below. 6. Stir the cellulose phosphate into 25 volumes of 0.5 M HCl. 7. Leave for 5 minutes. 8. Filter or decant the supernatant. 9. Wash the medium (with distilled water?) until the pH is above 3.0.

Procedure: Equilibration of cellulose phosphate

1. Immediately transfer the cellulose phosphate into 20+ volumes of concentrated buffer solution. 2. Stir the mixture. 3. Titrate the slurry with the base component of the concentrated buffer to the correct pH. 4. Filter or decant the supernatant. 5. Stir the cellulose phosphate into 20 volumes of the starting elution buffer. 6. Leave for 3 - 4 minutes. 7. Repeat steps 4 and 5 until the filtrate of the supernatant has the same pH and conductivity as the starting buffer.

Procedure: Binding and enzyme assay

1. Add 20mL of washed phosphocellulose to the homogenate, and stir continuously for 1 hour. 2. At time intervals of 10 minutes 0.5 ml aliquots from the stirring sample are to be removed (10, 20, 30, 40, 50, 60 minutes) for enzyme assay. a. Centrifuge aliquot 3 minutes (briefly), after this remove the supernatant. b. Add .025mL of the supernatant to a quartz cuvette, fill to 1mL with assay solution. c. Monitor absorption at 290 nm using UV/Vis. the data that needs collected is changer in absorption per minute. Which will be used with formula in the student hand out to determine the activity. d. Repeat for all 6 Sample aliquots taken. 3. Allow the phosphocellulose mixture to now settle for 15 minutes. 4. Remove a 0.5mL aliquot of the supernatant present; this will be used for an enzyme assay. 5. Vacuum filter the slurry of phosphocellulose using a 500ml Buchner funnel, with #4 Filter Paper. 6. Successively wash the phosphocellulose with two 100ml portions of extraction buffer, followed by two washing with MES-Tris buffer. a. Ensure phosphocellulose never goes to dryness. 7. Record the Volume of the combined washes (625mL). 8. Remove a .5mL aliquot for enzyme activity assay. 9. Create a slurry of phosphoscellulose with some of the MES-Tris Wash buffer. 10. Placed slurry into flask for later testing. 11. Ask Andreas what’s next.

Preparation of 4 L of MES-Tris Wash Buffer for Dialysis

1. Weighed out 67.017 g of KCl and placed into a 1000 mL beaker labeled "Beaker A". 2. Weighed out 67.004 g of KCl and placed into a 1000 mL beaker labeled "Beaker B". 3. Weighed out 9.770 g of MES and placed it into "Beaker A". 4. Weighed out 9.715 g of MES and placed it into "Beaker B". 5. Added approximately 800 mL of water to "Beaker A". 6. Added approximetely 800 mL of water to "Beaker B". 7. Added a stir bar to each beaker. 8. Placed the two beakers on two stir plates. 9. Calibrated a pH probe using solutions with a pH of 4.01 and 6.86. 10. Slowly added solid tris to "Beaker A" until the pH reached 6.5. 11. Slowly added solid tris to "Beaker B" until the pH reached 6.5. 12. Transferred the contents of "Beaker A" into a 2000 mL volumetric flask and labeled the flask. 13. Transferred the contents of "Beaker B" into a 2000 mL volumetric flask and labeled the flask. 14. Added 140 uL of 2-mercaptoethanol to each flask. 15. Stored the flasks in ice baths.

Washing phosphocellulose

The phosphocellulose was added to a column and eluted with MES-Tris, until the absorbance of the eluant was less than 0.01 on the UV/Vis at 280nm (ABS=0.002). The phosphocellulose was then eluted with potassium pyrophosphate, 4-ml samples were extracted and the absorbance tested at 290nm wavelength on the UV/Vis. The absorbance was recorded for each sample (See graph Absorbance of Protein Samples below). It was determined that the 15th and 16th samples contained the enzyme.

Determination of Enzyme Activity and concentration

3.975 ml of potassium pyrophosphate was added to a cuvette, and 25 microliters of the sample was pipetted on the rim of the cuvette. The contents of the cuvette were mixed together and quickly analyzed with the UV/Vis to determine enzyme activity. The activity was calculated by plotting the absorbance of the contents of the cuvette over time (60 seconds), and determining the slope of the curve when plotted on a graph; the slope is the activity.

The results of the enzyme activity for sample 15, sample 16, original "chicken soup," post-wash chicken, 30-minutes, and 60-minutes centrifuged chicken were recorded.

  • Sample 15:0.0069
  • Sample 16:0.0057
  • Original:0.0315
  • 30-Wash:no activity*
  • 60-Wash:no activity*
  • Post Wash: 0.0081
  • NOTE: no activity = slope of less than zero

BCA ASSAY

1. Added 25.05 mL of Reagent A (bicinchoniinic acid solution) to a 50 mL volumetric flask. 2. Added 0.50 mL of reagent B (copper(II) sulfate solution) to the flask. 3. Swirled the flask to mix. The solution turned bright green. This is the working reagent. 4. Added 2 mL of working reagent to each of 9 test tubes. 5. Added MES-Tris buffer and protein standard to the test tubes as shown in the table below. 6. The test tubes were labeled and placed in a warm water bath for approximately 30 minutes. 7. Measured the absorbance of the samples at 560 nm using the Spec-20. The results are shown in the table below.

Image:BCA.jpg

SDS-Page

1. Added 5 uL of 1xSDS to each of 6 microtubes. 2. Added 1 uL of BPB to each microtube. 3. Added 2 uL of 2-ME to each microtube. 4. Added 41 uL of each sample to seperate microtubes. 5. Labeled the microtubes (15-1, 15-2, 16, O, W, and F1). 6. The microtubes were centrifuged to remove the dye from the sides. 7. Placed the microtubes into a hot water bath for a few minutes. 8. Drew wells on the gel using a Sharpie (drew on the side with tape). 9. Took the tape off of the gel. 10. Put the gel into the electrophoresis unit. 11. Filled the middle part of the electrophoresis unit with 10xSDS triglycine buffer. 12. Poured the rest of the buffer into the sides. 13. Flushed the wells by squirting some buffer into them. 14. Loaded 10 uL of the standard size marker into the end well. 15. Loaded approximately 10 uL of each sample into 6 seperate wells. 16. Loaded Lemley solution into the last 3 empty wells. 17. Hooked up the electrophoresis unit. 18. Packed the electrophoresis unit in ice. 19. Ran the samples at 200 V for 30 minutes. 20. After 30 minutes, the electrophoresis unit was turned off. 21. The gel was removed. 22. The gel holder was broke open. 23. The gel was placed in a small pan filled with distilled water. 24. The gel was allowed to soak for 5 minutes. 25. The water was poured off. 26. Coomassie G250 stain was poured on the gel (just enough to cover the gel).

Calibrated Molecular Weights of Kelidoscope Standards

Image:Table.jpg

Chart: Absorbance of Fractions @ 280nm

Image:Absorbance of Fractions.JPG

SDS Page Results

Here is just a scan of the gel print-outs. I will try to find the time to get better files to you later. But I believe this should be sufficient for you write-up. Image:SDSPAGE0001.jpg

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