CERTL Program - Week 5

Goal: To observe the reactions of cytochrome P-450, be able to understand chemical binding, be able to read a binding chart, and be able to understand HPLC charts.

Activity 1 - Creating a Cytochrome P-450 Reaction, Part 1
The first thing you will need before performing this is a background of cytochrome P-450. Cytochrome P-450 is an enzyme in your body that metabolizes objects, changing them from one compound to a smaller compound called a metabolite. It can be both an enemy and a friend. As an enemy, it can make a lipophilic (fat liking) compound hydrophilic (water liking, and water soluble). It can also be a friend by making some fatal drugs/compounds, such as seldane, into something that can help the body, like allegra. One reason for performing this test is to see how it reacts to a drug. Because there are many types of P-450, four prototype drugs were used since each of them had a different P-450 Isoform.
Begin by creating a spreadsheet. The spreadsheet will help show how much of each ingredient will be in each microfuge tube. You will have several ingredients: protein, NADPH, Magnesium Chloride (MgCl2), Potassium Phosphate buffer (KPO4), and water (H2O); all of these ingredients would total 200uL per tube. All tubes had 8uL MgCl2, 40uL KPO4, and 25 uL NADPH (which we accidentally wrote as 5 on our spreadsheet and changed it after the spreadsheet was printed). You will also use 15uL of rat kidney tissue in select tubes, 29uL of liver tissue, and 63uL of lung tissue. Water will also be applied to give the tubes a total volume of 200uL. One tube with each tissue, along with a doublecheck for the three, make a set (total of six tubes). You can also see a simple synopsis of this in Week 3, Activity 4. There are three sets in the time dependence test, all of which depend on this setup. There are also four sets for the protein dependence test, which use different tissue setups. The first set has (all numbers in microliters): Kidney, 5; Liver, 10; and Lung, 20. The second set is (in microliters) 10 for Kidney, 20 for Liver, and 40 for lung. The third set will be the same as a time dependence set. The fourth set, though, has (in microliters) 20 Kidney tissue, 40 Liver tissue, and 80 Lung tissue. Next to each set include a time. All of the protein dependence tests are 20 minutes and each of the time dependence tests are assigned 10 minutes, 20 minutes, or 40 minutes (choose one for each).
Begin the experiment by removing the evaporated drugs that were made from Week 3, Activity 4 from the freezer. Add the desired amount of tissue into each microfuge tube. You must put the tube rack(s) on ice once you have put all of the tissue into the tubes. While the tubes are on ice, you may add the MgCl2, H2O, and KPO4 in any order desired (the NADPH is done later). Make sure there is an incubator ready for the next part, for now you will be putting the tubes into the incubator in a specific order. It may be wise to put your tubes in the freezer and prepare a chart with when each tube will go. The chart has three columns: tube, time in, and time out. The first tube that goes in will be at 0:00 (o min., 0 sec.), and each tube following will be thirty seconds after the previous. The time out will be the tubes time in time + the time assigned to it on the spreadsheet. You may have to do your time and protein dependence tests separately due to this. When you're ready, take the tubes for whichever test you will be performing and place them next to the incubator. Also set up a pipetter to put the needed amount of NADPH in the tube. Before the tube goes in, it must have the NADPH inserted and have been vortexed for a small amount of time. The microfuge tubes are otherwise put in at the time specified by the sheet. It is best to do with a partner, who can look at a stopwatch and give you the tubes at the appropriate time while you put in the NADPH and vortex. When it is also time, take out the tubes and put in the freezer. Unfortunately, we ran out of time afterwards to finish and our mentor did the rest for us. He said we did not get the appropriate results, anyway, and a new one was done. This will be seen later in the post as "Creating a Cytochrome P-450 Reaction, Part 2."

Activity 2 - Chemical Binding
This experiment allows you to develop an IC50 for a compound/drug. You will begin with two tube racks on ice. In the first rack, you will setup nine rows of three scintillation tubes. The rows are labelled N (short for nonspecific), T (short for total binding), 10, 30, 10^2, 3e2, 10^3, 3e3, and 10^4. The second rack has seven rows of three scintillation tubes. These all have the same labels as the first rack except that there are no N or T rows. You will also need to make four additional tubes (on the side) labelled 10^2, 10^3, 10^4, and 10^5. I will add an x to the end of the side tube labels so that you do not confuse them with the tubes on the rack. Put 200uL of your compound/drug and 1800 of your buffer into 10^5x tube. Vortex this tube and put 20uL of it into the 10^4x tube. Add 1980uL of buffer to all of your side tubes except 10^5x. Vortex the 10^4x tube and put 20uL of it into the 10^3x tube. Vortex the 10^3x tube, put 20uL of it into the 10^2x tube, and vortex the 10^2x scintillation tube. A chart below is given with how much of each ingredient must be added to which tubes (the rows that are mentioned include all of the scintillation tubes on the row). You will be unable to do the tissue (we used rat stratium) until it is thawed and homogenized. The N row uses a different drug than the other rows. We used WF23.

You will now place all of the tubes in one rack with the rows going from right to left, starting with rack 1's N row and finishing with rack 2's 10^4 row. The four side tubes are not included in this new setup. The rack is run through a Brandell. The radioactivity is then counted and the rack's data is evaluated.

Activity 3 - Reading Chemical Binding Charts

When you are done with the binding, a chart will be produced with results. You will use this and a special pre-made spreadsheet to calculate the IC50 of your drug/compound. The first thing you will have to do is look at the chart's coefficient of variances (sometimes abbreviated COEF. OF VAR) and note which ones are greater than 10. For the sets that are greater than 10, look at their 3H DPM, eliminate the number that is the farthest from the others, and re-average the remaining two numbers (this will take the place of the Replicate Average DPM for 3H). The next thing to be done is look at the Replicate Average DPM for 3H's and see if they are between the Replicate Average DPM for 3H's of N and T (set 1 and2). If they are not between these numbers, then the set is eliminated. The remaining Replicate Averages are added to the appropriate slots on the spreadsheet and an IC50 is given.

Activity 4 - Creating a Cytochrome P-450 Reaction, Part 2

The next try had some new measurements, fewer tubes, and a partially different process. There will be two sets of six microfuge tubes, one with 30 uL of kidney tissue in each tube, and one with 30uL of liver tissue in each tube. In the spreadsheet, all of the tubes will have 10uL of KPO4 buffer and 2uL of MgCl2. We also decided to see how our compound would be affected by Cytochrome P-450 (friend or enemy); therefore, each set had two tubes with our prototype drug, two with our compound, and two with both drug and compound (one high concentration and one low concentration). There would be one prototype drug without NADPH, one compound without NADPH, and both combined tubes had NADPH. The high concentration has 5uL of our prototype drug and 5uL of our compound, the low concentration has 5uL of our prototype drug and 2uL of our compound. On the spreadsheet, the tubes with NADPH get 175uL of water and 25uL of NADPH while the tubes without NADPH get 200uL of water. You are now ready to begin. You will have to evaporate your drug first, and then add the tissue. The microfuge tubes are then put on ice where you will insert (in this order): KPO4 buffer, MgCl2, and water. The tubes that need NADPH are given NADPH right before you put the whole rack of tubes in the incubator for one hour (be sure to vortex after giving them NADPH). After one hour, the rack is removed and put in the freezer (to stop the reaction) until you are ready for the next step. When you are ready, remove the tubes from the freezer, add 300uL of ACN HCOOH to each tube (this separates the solution from the protein), vortex vigorously for one minute (I had it on 3000 for speed), and put in a microfuge centrifuge at 8000 rpm for fifteen minutes. When the tubes are removed from the centrifuge, you will transfer the liquid (leave the pellet behind) into a vial for an HPLC.

Activity 5 - Reading an HPLC Chart

An HPLC chart is very simple, there should only be one peak. The later it occurs on a line, the longer it took for the compound to come off of the column, also known as retention time. The peak height also determines the concentration of the compound. If there are any other smaller peaks, then there may be another compound on the column.

Originally posted: July 15, 2005 9:58 AM

Edited and re-posted: July 17, 2005 12:42 PM

CERTL program - Week 4

Goal: To understand NMR, column chromatography, and tissue buffers.

Activity 1 - Column Chromatography
Column chromatography, unlike thin layer chromatography (TLC), is quantitative and able to separate compounds. We needed to perform a column chromatography because our TLC showed two compounds in our solution. There are two ways you can do this, one with fancy machinery (including a computer) and one with a glass column. We used a glass column and will explain this one to you. A picture below shows a glass column and a TLC (I slighty edited this web picture so that you can see which is pointed to which).
Clamp a column over an Erlenmeyer flask and stuff glass cotton into the bottom without clogging up the hole. Pour powdered silica into the column through a funnel followed by enough 1:1 (hexane:ethyl acetate, you use what is one less polar to finding your two blots) to wet everything. Blow compressed air into the column to elimate any bubbles, but not too much or else your liquid will pour out too quickly. Clean the top by sucking in and out (pressing and releasing) with a pipette. Allow more compressed air to blow into the column and allow some drops to enter your solution. Continue blowing compressed air until the silica and liquid level are almost even (the liquid slightly higher) and pour in your solution. Add some more 1:1 (or whichever solvent is needed) so that nothing dries. When enough solvent is poured, you may blow compressed air and begin filling your test tubes. As the tubes are being filled, perform TLC's (see week 2, activity3) on them to find which ones contain your compound. Combine the tubes with the same compound together and the tubes with no compounds together. A "flush" is then performed by filling the column with ethyl acetate and pouring it into a round bottom flask to search for more compound. The three sets are then rotary evaporated and separation can be checked by a TLC.

Activity 2 - Preparing For A Nuclear Magnetic Resonance Spectroscopy (NMR)
An NMR must be done in specific labs where an extremely strong magnet is used (see below), and the solution must be prepared beforehand. To prepare, pipette a small amount of your solution into a long, thin container. Then pipette some chloroform into the container. The container is to be capped and labeled with your name, date, chemical structure, and chemical name.









Activity 3 - Reading NMR
When you get to the lab with your sample, it will depend on the machine and program used to perform your NMR. The graph produced (see below), though, is the same. To determine what your compound should look like, you must know its structure. Note where each group of hydrogens is, and compare them to their neighboring groups, add 1 (the n+1 rule) to determine how many peaks there will be. Check your graph for the appropriate peaks and ratios (a Pascal's triangle can be used). If more peaks are present, then your compound is not yet pure and other compounds are present. For more information and most probably a better explanation, go to http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/nmr1.htm.

Activity 4 - Making Tissue Slice Buffers
We will be dissecting a rat soon, and we will need some buffers to keep its cells alive. A buffer is a chemical or enzyme that usually weakens or strengthens a chemicals pH level so that it does not harm an organisms body. We began by making a stock that would be used in all three of our buffers. Pour 200mL of water into a beaker with a magnetic stirrer and put it on a hot plate. Only turn on the hot plates stir function and begin weighing (as well as pouring into the beaker) the following amounts of ingredients:
Potassium Dihydrogen Phosphate- 0.0326 g
Sodium Bicarbonate- 0.42 g
Magnesium Chloride- 0.0286 g
Calcium Chloride- 0.0441 g
Glucose- 0.36 g
Allow this to stir until all of the ingredients have dissolved. While this is happening, label three more beakers with the following titles: "50 mL potassium," "100 mL incubator," and "50 mL pre-incubator." When the stock is done, pour 100 mL into the "100 mL incubator" beaker, 50 mL into the "50 mL potassium" beaker, and 50 mL into the "50 mL pre-incubator" beaker. Now each of these are stirred while adding some more ingredients to each beaker. Below are the ingredients for each beaker.
50 mL potassium
Potassium Chloride- 0.112 g
Sodium Chloride- 0.319 g
Ascorbic Acid- 0.0018 g
100 mL incubator
Potassium Chloride- 0.0358 g
Sodium Chloride- 0.7 g
Ascorbic Acid- 0.0035 g
50 mL pre-incubator
Potassium Chloride- 0.0179 g
Sodium Chloride- 0.351 g
When they are done stirring, store them in a safe place so that they are not spilled.

CERTL Program, Week 3

Goal: To find a drug's metabolite and teach students what we've learned

Activity 1 - Creating calibration charts
A repeat of activity 3 from week 1 is done with a slight difference. 100 uL of the 1:5, 1:10, and 1:20 solutions are put in a microfuge tube, along with 200 uL of water to change the solutions to 1:15, 1:30, and 1:60 ratios. These tubes are vortexed and have 10 uL of each solution placed in a well of a 96-well tray. 10 uL of the nine calibration samples and double checks for each are placed in the tray, filling 36 wells. 150 uL of Bradford reagent is then applied to each well with a solution in it. We used a special pipette that holds 250 mL of reagent and sprays 150 uL of reagent with each click. The tray is read in the Tecan GENios™ and only the cells that correspond to wells with solution are used. The original and double check of each calibration are averaged and lined up with their concentration. A scatter plot can be formed from this data. To see if the line is linear, obtain the R2 value by right clicking on a point and adding a trendline. The best is a 1.0. I was close, my graph is shown to your right below. Also obtain the equation (it will be in y=mx+b), which is used with the average of the original and double checked organ solutions (y) to obtain the concentration (x).

Activity 2 - Making aspirin
A summer camp came and we assisted our mentor with helping the students make aspirin. They poured 1g of salycylic acid, 2 mL of acetic anhydride, and 3 drops of phosphoric acid into an Erlenmeyer flask. The ingredients were mixed and heated on a hot plate for 10 minutes. Ten drops of distilled water is added to the heated mixture, followed by 10 mL of distilled water. It is placed in an ice bath until most of the solution has crystalized. When crystalized, a sheet of filter paper is folded into sixteen equal pieces (to increase surface area) and placed inside a funnel connected to a clean Erlenmeyer flask. The crystalized solution is poured into this and gravity will remove excess water and any vinegar that is made (the equation is salycylic acid + acetic anhydride + phosphoric acid --> aspirin + vinegar). When only crystals remained, the students weighed what they made and took it home in a container.

Activity 3 - Removing caffeine from tea
The summer camp returned and we helped our mentor with helping the students extract caffeine from tea. Before anything scientific happened, they made some tea. It was poured into an Erlenmeyer flask and 15 mL of dichloromethane was added. It was shaken up and the bottom layer (the dichloromethane) was removed from it by pipette. We would have used seperatory funnels (like in week 2), but there were not enough. This was repeated two more times so that the caffeine (which would attach to the dichloromethane) was removed from the tea, allowing the tea to be poured out. The three extractions of dichloromethane are placed in a beaker with 20 mL cold (we made it that morning before the students came, and trust me, it burned my hands just shaking it) 6 molar sodium hydroxide. It was shaken and then the top layer (the sodium hydroxide) was removed. The same thing is done again, but with 20 mL of cold distilled water, it is the removed top layer. Magnesium sulfate is put in the solution until it snows (showing no more water) and poured into a beaker (leaving the magnesium sulfate behind). This beaker is put on a hot plate until all of the liquid is gone. A powder will be on the bottom of the beaker, which is the caffeine. The students weighed what they made and put it in a container to take home.

Activity 4 - Preparing to test how long it takes for a drug's metabolite to appear
To prepare, we learned about cytochrome P-450, which digests drugs and makes them water soluble (lipophilic to hydrophilic). We then were assigned different drugs, mine was S-Ibuprofen. To prepare, a buffer is made by pouring 350 mL of water in a beaker, 31.4 mL of phosphoric acid (ALWAYS add acid to water), and filling the graduated cylinder that had the phosphoric acid a little bit at a time and pouring into the beaker until the solution is 500 mL. Our plan is made on a spreadsheet so that we know how much of each chemical/ingredient we need. Since 42 test tubes are needed, each set at a different time of checking (each set contains six tubes, containing the three organs and a double check). The chosen drug is placed in each microfuge tube (the amount depends on the equation Volume1 times Concentration1 = Volume2 times Concentration2) and placed in the oven at 35 degrees Celcius until the liquid has evaporated and only the drug remains. Place this in the freezer until you are ready to use it.

CERTL program, Week 1

Goal: To understand the procedure for testing drugs.

Activity 1 - Preparing stock solutions
The experiment began with a semi-reproduction of an experiment conducted by Kenneth O. Ebete and John E. E. Koundourellis. We made stock solutions with Diphenylpyraline (DPP) just as they did, but with different amounts. DPP is an antihistimine composed of two benzene rings and one piperidine ring with the chemical formula C19H23NO·HCl. Its chemical structure is shown below:
The abstract of their experiment can be seen at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8704936&query_hl=1.
The stock solution used had a ratio of 1mg/mL (DPP/methanol). The 999 mL of methanol was used to dilute the solution and make it maintain a volume of one milliliter. This solution was then used for eight "small, screw top test tubes," each with a volume of 250 mL. This reduced the ratio by one fourth, converting it into 250 ng/250 mL. The new solutions were then made made with a linear amount of stock solution (y=mx+b) and diluted with methanol for a volume of 250 mL. I used a slope of 15 for my solutions.

Activity 2 - Collecting Endoplasmic Reticulum (ER)
ER is an organelle in a cell that allows the transportation of materials throughout the cell, similar to a highway. With permission, ours were obtained through rat liver, kidney, and lung. The process started in a cold room, where the the temperature was at 4º C. In the cold room, the liver, kidney, and lung were homogenized (broken down into just cells) cells), creating a liquid-like substance. After evenly pouring them into test tubes, a buffer was inserted so that all of the test tubes tubes would weigh as close to 13.00 grams as possible. Afterwards, the tubes were placed into a centrifuge (an example of one is shown below) for differential centrifugation


a centrifuge

Differential centrifugation is composed of several steps. The first one is spinning at 3000 xg for 10 minutes. This separates the the nuclei and membrane from the tissue and forms a lump in the bottom of the test tube known of as a pellet. It is then spun at 9000 xg for 20 minutes, adding mitochondria to the pellet. The remaining tissue is then transferred into a clean test tube, leaving leaving the pellet behind. The new tube is then spun in the centrifuge for 45 minutes at 35000xg, which separates the ER from the tissue. The tissue is then frozen with the ER pellet.

Activity 3 - BPA (Bradford Protein Assay)
The frozen test tubes from activity 2 are remixed (the tissue and the ER pellet). A protein water was then made with a ratio of 5mg/mL, using water for dilution. After this, eighteen concentrations were made. The first nine were using the protein water and diluting it with clean water (creating a secondary stock solution) for a total volume of one mL. The other nine were using the the tissue and and diluting it with clean water at ratios of 1:5, 1:10, and 1:20 (tissue to overall volume) to create a volume of one mL. All eighteen solutions are placed twice (to double check results) on a 96-well tray (filling 36 of the holes). Bradford reagent was was then added to the wells that contained a solution. After incubating for 30 minutes, they were inserted into an object made by Tecan called a GENios™ (a picture from their website is shown below), where the absorbance of light was measured, and results were provided.

CERTL Program, Week 2

Goal: To create chemical compounds and understand compound tables.

Activity 1 - Compound Tables
Compound tables are a list of compounds that will be used. Besides their name, equivalent, their molecular weight, weight weight in moles, weight in grams, density, and volume in milliliters will be provided. Equivalents are ratios of one compound to another, this will be used to determine the moles of compounds. To get the moles, you must know how many grams of one compound will be used. Divide this by the corresponding compound's molecular weight. The equivalent ratios are then made to determine the moles of the remaining compounds. Multiply these by their corresponding compound's molecular weight to get the amount of grams used for them. If the compound is a liquid, density and volume will be needed. The density is a gram to mL ratio. Apply this ratio to get the volume of the compound. An example is provided below for a visual example that when used right, can make aspirin and vinegar. H3PO4 is only used for three drops, which is why most of its data is not given.






Activity 2 - Creating chemical compounds
Begin by creating a compound table for all of the chemicals that will be needed, as well as the product. After the table has has been made, collect the appropriate grams or milliliters of each compound (except for the product) and put them in a flask. If the compounds must be heated and stirred to mix, place the flask on a heat plate with a magnetic stirrer and set the heat and stir to needed levels. Be sure to attach a condenser to the flask in order to catch and return water vapors. When the compounds are mixed, they are quenched with 5 mL of water and 10 drops of HCl. Apply this to a separatory funnel and add 40 mL of ether. Now several chemicals will be added, shaked, vented (open hole away from faces) to release pressure), and poured (only the bottom layer, you can see a line that separates them). The chemicals, in order, are 10 mL water, 40 mL water, another 40 mL water, 40mL sodium bicarbonate, and 40 mL sodium chloride. After each chemical, also apply a backwash (pour the remaining layer into another flask; repour the chemical; shake, vent, and pour; and repour the layer that you put in another funnel). When done with all of the chemicals, put in a flask with magnesium sulfate. Shake and add magnesium sulfate until it stops clumping and appears to be snowing inside the solution. Pour this into a flask connected to a dryer through the dryer to eliminate the magnesium sulfate. All that should remain is the solution and ether. To eliminate the ether, the remaining solution is placed in a rotary evaporator.

a rotary evaporator

It is started by attaching a round bottom flask to the machine. Following the attachment, it is lowered into a tub of water at a specified number of degrees (about 40ºC). The rotator is then turned on so that the solution will spin and increase pressure. To contain the pressure, make sure that the rotary evaporator is connected to a sink with both hot and cold water on at full power, and the vacuum at the end is sealed. Watch your flask to ensure that the solution does not suddenly shoot up (if it does, stop the process at once). When the solution changes color and its level does not seem to change, it is complete (and, if done right, pure). Weigh the object you will store the solution in, pour in the solution, weigh the combination, and subtract the two for the weight (amount)of your solution. The you should have according to your table is known as theoretical yield. The amount you actually have compared to your theoretical yield percentage wise is the percent yield.

Activity 3 - Performing Thin Layer Chromotagraphy (TLC)
This process is very simple and will provide an Rf value and how complete your solution is. To begin, cut a rectangular silica strip and place a light pencil dot near the bottom of it. Take some of your compound through a capillary and blot it on the dot, let it dry. While this dries create a solution of hexane to ethyl acetate (the ratio depends on how polar it needs to be), this will be the solvent. Pour a very small amount of your solvent into a beaker (it should not be as high up as the dot on your silica strip) and place your strip into the beaker vertically, with the blot on the bottom. Capillarity will cause the solvent to climb the strip and carry your solution with it for some time. When the solvent is near the top, remove the strip, lightly mark where the solvent stopped on the strip, place under a UV light, and mark where the blot is. To determine the Rf value, divide the distance from the dot to the blot by the distance from the dot to the line. If a substance is being dissolved and you want to see whether it has dissolved yet or not, you can perform a TLC with both on the same strip (one on the left and one on the right) and see if the blot is in the same place (showing the substance is still present).