Biochem Lab 385 Fall 2009 Instructor Heather Kundzicz ION by reuotld5

VIEWS: 0 PAGES: 6

									BIOCHEM LAB: ION EXCHANGE CHROMATOGRAPHY

FALL 2009

Biochem Lab 385 Fall 2009 Instructor: Heather Kundzicz ION-EXCHANGE CHROMATOGRAPHY INTRODUCTION: Ion exchange chromatography is a method of purifying molecules based on their ionic charge. It is a useful technique for separation of proteins, because proteins are either multivalent anions or cations. The ionic charge of a protein depends on its amino acids and their side chains, amino groups and carboxyl groups. Some basic amino acids (negative charge), for example, are lysine, arginine and histidine, and acidic amino acids (positive charge) are glutamate, aspartate, cysteine and tyrosine. Since many amino acids make up each protein, then each protein has an isoelectric point, or pI, which is the pH where the protein has a net zero charge. Thus, the charge on a protein can depend on the pH of its environment. For example, at high pH, carboxyl groups are deprotonated, and the amino groups become free bases: R-COOH R-COO R-NH3+ R-NH2

such that the protein would take on a net negative charge. So, if the pH > pI the protein loses protons to the buffer and becomes negatively charged. If pH < pI the protein gains protons from the buffer and becomes positively charged. Therefore, all proteins are charged except at their pI. Ion exchange chromatography uses this property of ionic charge to separate proteins on a matrix that has an opposite charge. Ionic groups are covalently bound to a stationary phase gel such as cellulose or dextran beads, or resin. With the right buffer, proteins can bind (or not) according to their charge. A stationary phase with a positive fixed charge would bind negative molecules, anions, and is called an anion exchanger. A stationary phase with a negative fixed charge would bind positive molecules, cations, and is called a cation exchanger. Proteins can then be eluted from the stationary phase with increasing ionic strength, or salt. Actually, the ions from the salt compete for charged sites on the solid phase, thus displacing a bound protein.

For example, a sample might contain two negatively charged proteins, one more negatively charged than the other. If this sample were applied to a positive, or anion exchanger, both proteins would bind under the proper conditions. The less negative protein would bind weakly and could be eluted from the column by increasing the ionic strength, or salt, in the buffer. Further increase in ionic strength of the elution buffer would then elute the second protein. Changes in pH might also be used for elution.
Rev 112508HK 1

BIOCHEM LAB: ION EXCHANGE CHROMATOGRAPHY

FALL 2009

The pH of the buffers used in a separation must be considered. Ideally the pH of the buffer should be slightly higher than the pI of a protein when performing anion exchange. In that way, the protein will tend to have a more (-) negative charge and will bind better to the (+) anion exchanger. Similarly, the pH of the buffer should be lower than the pI of the protein when performing cation exchange. At the same time, the pH of the buffer must be high enough (or low enough) to keep a net negative (positive) charge on the cationic (anionic) matrix. We will be using two ion exchangers in this lab, a cationic exchanger Phosphocellulose, which has a negative phospho group bound to a cellulose bead matrix, and an anionic exchanger DEAE-cellulose, which has a positive diethylaminoethyl group. Other cationic exchangers might include carboxymethyl cellulose (CM-cellulose), or sulfopropyl dextran (SP-Sephadex). Other anionic exchangers include polyethyleneimine cellulose (PEI-cellulose) or diethyl- (2hydroxypropyl)-aminoethyl (QAE-Sephadex). We will apply 2 proteins to the ion exchange columns, and determine their elution profile via protein estimation by the Biorad micro-method, which is a qualitative interpretation of protein concentration.

MATERIALS AND METHODS: Prepared phosphocellulose (PC) resin, cation exchanger* Prepared DEAE-cellulose (DEAE) resin, anion exchanger* 4 Bio-Rad Mini-columns; 2 containing PC and 2 containing DEAE Microcentrifuge tubes Test tubes Elution Buffer A (50 mM Tris-HCl, pH 7.5-8.0 + 0.05 M KCl) Elution Buffer B (50 mM Tris-HCl, pH 7.5-8.0 + 0.30 M KCl). Elution Buffer C (50 mM Tris-HCl, pH 7.5-8.0 + 0.60 M KCl). Elution Buffer D (50 mM Tris-HCl, pH 7.5-8.0 + 1.00 M KCl). BSA = Bovine serum albumin, 0.4 mg/ml in Buffer A, (pI = 4.8) CytC = Cytochrome C, 0.4 mg/ml in Buffer A, (pI = 10.6) BSA and CytC positive controls for protein testing. Micropipettors Biorad protein assay reagent, Diluted 1:5 with dH20 Microtiter plate Centrifuge pH test strips, cut in half length-wise. **Preparation of ion-exchange media: (already done for you courtesy of Dr. Ackerman) Phosphocellulose and DEAE-cellulose were prepared from powders. The powders were resuspended as follows. Phosphocellulose: 50 grams of powder were resuspended in 1000 ml of 0.5N NaOH. This slurry
Rev 112508HK 2

BIOCHEM LAB: ION EXCHANGE CHROMATOGRAPHY

FALL 2009

was stirred for 30 min, and then filtered through a Buchner filtration apparatus. This process takes at least 15 min. The resin was washed with water on the suction filtration until the pH was that of the water. The resin was then placed into 1000 ml of 0.5 N HCl. The resin was stirred for 30 min and then filtered as above. The resin was washed with water until the pH was the same as the water. DEAE-cellulose: 50 grams of powder were resuspended in 1000 ml of 0.5 N HCl. This slurry was stirred for 30 min, and then filtered through a Buchner filtration apparatus. This process takes at least 15 min. The resin was washed with water on the suction filtration until the pH was that of the water. The resin was then placed into 1000 ml of 0.5 N NaOH. The resin was stirred for 30 min and then filtered as above. The resin was washed with water until the pH was the same as the water. The washed resins were each resuspended in water containing 0.02% Na azide, and stored at 4oC. EXPERIMENTAL PROCEDURES A. Ion Exchange Procedure 1. You will be provided with 4 mini-columns: 2 contain phosphocellulose (PC) and 2 contain DEAE cellulose. The PC and DEAE are equilibrated in Elution Buffer A. You will received them in tubes labeled as in row #1 below. Plan carefully! Loss of even one drop during collection might ruin your results! 2. Label 16 microcentrifuge tubes (4 sets of 4) as follows in rows 2-5. If you use 4 color-coded tubes it will be easier to keep track of your samples.
Color used For PC#1________ 1 PC#1, 0.05M KCl For PC#2 ________ PC#2, 0.05M KCl For DEAE#1 _________ DEAE#1, 0.05M KCl For DEAE#2_________ DEAE#2, 0.05M KCl DEAE#2, CytC, 0.05M KCl DEAE#2, CytC, 0.3 M KCl DEAE#2, CytC, 0.6 M KCl DEAE#2, CytC, 1.0M KCl

2 PC#1, BSA, 0.05M KCl PC#2, CytC, 0.05M KCl DEAE#1. BSA, 0.05M KCl 3 PC#1, BSA, 0.3M KCl PC#2, CytC, 0.3 M KCl 4 PC#1, BSA, 0.6M KCl PC#2, CytC, 0.6 M KCl 5 PC#1, BSA, 1.0M KCl PC#2, CytC, 1.0M KCl DEAE#1. BSA, 0.3 M KCl DEAE#1. BSA, 0.6 M KCl DEAE#1. BSA, 1.0M KCl

3. Remove the yellow column caps from the bottom of each column; save caps for end of lab. Each mini-column should be in the appropriate microcentrifuge tube: PCmini column #1 tube PC#1, 0.05M KCl PC mini column #2 tube PC#2, 0.05M KCl DEAE mini column #1 tube DEAE#1, 0.05M KCl DEAE mini column #2 tube DEAE#2, 0.05M KCl 4. Leave top caps on while centrifuging. Place the mini-column/microcentrifuge tubes in the centrifuge and spin as directed by the instructor. Please note the centrifugation procedure for your report.
Rev 112508HK 3

BIOCHEM LAB: ION EXCHANGE CHROMATOGRAPHY

FALL 2009

5. Remove the mini-columns and place them into the NEXT set of microcentrifuge tubes for keeping until step 7, then go to Step 6. PCmini column #1 PC mini column #2 DEAE mini column #1 DEAE mini column #2
tube

PC#1, BSA, 0.05M KCl tube PC#2, CytC, 0.05M KCl tube DEAE#1, BSA, 0.05M KCl tube DEAE#2, CytC, 0.05M KCl

6. TESTING INITIAL CONDITIONS: The buffer and any components that did not bind to the gels in the mini-columns will be collected in the microcentrifuge tubes, and are to be saved for testing. The first eluant sample, from steps 3 and 4, is your negative control. There should be no protein in any of these first samples and the pH should be 7.5-8.0. Test as follows: a) Cap and mix each eluant sample collected from each mini-column in steps 3 and 4.. b) Test the pH of Elution Buffer A , and the eluant from each column: Pipet 20 ul of the buffer or each eluant sample onto a separate pH test strip. Record in data table under Negative Controls. c) Test for the presence of protein in the Positive Protein controls (BSA and Cyt C controls provided by instructor), Elution Buffer A, and the eluant from each column using the Bio-Rad Microtiter Protein Assay described below. (Total 7 wells in this part) d) Record all results in data table under Positive Controls and Negative Controls. ______________________________________________________________________________ Bio-Rad Microtiter Protein Assay 1. Pipet 150ul of the diluted Bio-Rad protein assay reagent into a microtiter plate well. 2. Mix the protein standard, buffer, or eluant sample to be tested. 3. Pipet 20ul of the buffer, protein standard, or eluant sample into the well. Mix by slowly pipetting up and down with the pipette. Place the microtiter plate on a white surface (not a paper towel) and observe the color change, if any, within each well. Interpret results as follows. Remember, this is a qualitative test and you are looking for the presence or absence of protein in the sample. A light blue reaction or a dark blue reaction are both positive! 0 = no color change + = light gray/faint blue within 10 minutes. ++ = light blue immediately +++ = dark blue immediately ______________________________________________________________________________
Rev 112508HK 4

BIOCHEM LAB: ION EXCHANGE CHROMATOGRAPHY

FALL 2009

LOADING PROTEIN ONTO THE COLUMNS and ELUTION OF PROTEINS: 7. The mini-columns are sitting in the next microcentrifuge tubes as directed in Step 5. a) Discard the Positive Protein controls and obtain the BSA or CytC Protein Samples. b) Remove the top column caps and add BSA or CytC Protein Sample to the mini-columns as follows: 50ul BSA Protein Sample 50ul CytC Protein Sample 50ul BSA Protein Sample 50ul CytC Protein Sample PCmini column #1 PCmini column #2 DEAEmini column #1 DEAEmini column #2

b) As soon as the 50ul of protein enters, or soaks into, the PC or DEAE, pipet 300ul Elution Buffer A into each mini-column. c) Replace the top caps* and centrifuge as directed. Remember to visually inspect each mini-column and eluant as you go…you may make some interesting observations! *Replace mini-column caps only when the columns are in the microcentrifuge tubes because when you put on the cap a drop of liquid may come out the bottom of the column and you don’t want to lose any sample!! 8. Transfer the mini-columns into the NEXT set of microcentrifuge tubes for keeping until Step 10, then go to Step 9. PCmini column #1
tube

PC#1, BSA, 0.3 M KCl, and so on!

9. Cap and mix the eluant from each column (tube PC#1, BSA, 0.05M KCl, etc.), then test the eluants for the presence of protein using the Bio-Rad Microtiter Protein Assay described earlier in the handout. You do not need to test pH again. Record in data table under Eluant Samples, 0.05M KCl. 10. Pipet 300 µl of Elution Buffer B (0.3 M KCl) into each of the mini-columns. Replace top caps and spin as directed. 11. Continue, placing mini-columns into next tubes, testing collected samples for protein, and adding 300 µl of the appropriate elution buffers (C, 0.6 M KCl and D, 1.0 M KCl respectively). ----------------12. When you are finished with the centrifugations and protein testing, please place the mini-columns in clean microcentrifuge tubes and pipet 300 µl of elution buffer D into each column and centrifuge. 13. Discard the eluant from the Buffer D wash. Wash/spin again with 300 µl of Buffer A and discard eluant. Pipet 300 µl of Buffer A onto each column, seal the columns top and bottom, and return columns to the instructors.
Rev 112508HK 5

BIOCHEM LAB: ION EXCHANGE CHROMATOGRAPHY

FALL 2009

Data/Results:
Positive Controls BSA Standard Cyt C Standard Negative Controls Buffer A (w/ 0.05MKCl) PC#1, 0.05M KCl PC#2, 0.05M KCl DEAE#1, 0.05M KCl DEAE#2, 0.05M KCl protein result protein result (bovine serum albumin) (Cytochrome C) pH

Protein in Eluant Samples 0.05 M PC, BSA PC, Cyt C DEAE, BSA DEAE, Cyt C Observations:

[KCl] 0.3 M 0.6 M 1.0 M

Not questions, just reminders of what to include in your intro/discussion/conclusion! Don’t forget to discuss the following topics within your introduction and/or discussion. Don’t forget to cite your references! Discuss how the experiment (should) work: Support your answer, specifically in terms of the isoelectric point for each protein tested, the pH and ionic strength of the buffers, and the ion- exchange matrix used. Compare your experimental results with the expected results. Discuss any discrepancies. Also include a brief description of the BioRad protein assay, as it was the detection method for your IEC experiment. And, of course, describe Cytochrome C and Bovine Serum Albumin, and their functions in vivo.

Rev 112508HK

6


								
To top