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Lab 6: Protein Purification

Jacob Feste

010617389

3/8/15

Objective

The objective of this experiment was to isolate a GST tagged protein Riboflavin Kinase (NcRFK) from bacterial cell lysate of which originally contained a plasmid, pGEX-KG-NcRFK, coding for the overexpression or production of the Riboflavin Kinase protein. In order to accomplish this, the bacterial cells containing the plasmid for the overexpression of NcRFK were lysed using ultrasonification, breaking the bacterial cells down into their different components. Affinity chromatography was then performed with this mixture of components using GSH-Agarose columns in attempt to separate and purify the components of the bacterial lysate. This method allows the mixture to separate its contents due to differences in properties such as charge, size, and hydrophobicity within the stationary phase of a culture. It then sends a mobile phase culture through the columns, allowing the mixture to separate and purify even more due to the differences in each components affinity for the stationary phase versus the mobile phase culture. Once purified by the affinity chromatography method, SDS-Page was then performed in order to identify the protein obtained and to isolate the protein even further to ensure the purification process didn’t leave contaminants. Isolation is due to size differences while running vertically with gel electrophoresis while identification is made possible by referencing the results to their expected molecular weights. After the affinity chromatography was performed, protein samples of bacterial cell lysate, flow through, and purified NcRFK were obtained and used in the SDS-Page experiment. Therefore the expected outcome of this experiment is separate bands for the different components of the protein sample as well as SDS-Page molecular weight readings similar to the expected molecular weight of NcRFK to provide that the NcRFK protein was successfully isolated.

Materials

1. GSH-Sepharose resin packed in a poly-prep column with ~2ml bed size.2. 1X PBS buffer; pH-7.43. 10 mM reduced Glutathione in 1X PBS4. 6M Guanidine hydrochloride solution5. 20% Ethanol in water6. SDS-PAGE gel and electrophoresis apparatus

Procedures

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Preparing the columns (Column packing):

1. Take ~1.5-2ml of GSH-Sepharose bead solution and pour onto the spin columns2. Twist open the bottom part of the column 3. Now transfer the freshly packed column into a 15ml centrifuge tube and place it on a

rack4. Let the storage solution drip (20% ethanol) into the centrifuge tube for 5 min allowing

the resin to settle 5. Add 2 ml of fresh 20% ethanol on top of the column and let it drip completely6. Close the bottom part of the column with an end cap 7. Add 1 ml 20% ethanol to the column 8. Now close the top part of the column with a lid and leave it in refrigerator till further

use

Purifying GST tagged proteins using GSH-Agarose column:

Each group of two will be provided with pre-packed column and all the required buffers

1. Take the column out of the 15ml centrifuge tube and remove the end cap (Do not discard the end cap. You will be needing it later)

2. Open the top lid and keep it aside along with the end cap.3. Keep the column back into the 15ml centrifuge tube and let the ethanol drip out for 5-

10min4. Discard the flow through solution5. Now add 5ml PBS to the column6. Leave the column in the centrifuge tube till all the PBS to drip out7. Discard the flow through solution8. Close the bottom part of the column with the end cap9. Add 1ml of bacterial soup to the column and leave it for 5 min10. Remove the end cap and let the bacterial solution drip out (This is the 1st unbound

fraction)11. Do not discard the solution – Transfer it into a 1.5ml micro-centrifuge tube and label it 12. Transfer the column into a new 15ml centrifuge tube – Discard the old one13. Add 5ml of PBS to the column and let it drip out (This is the 2nd unbound fraction)14. Transfer some of the solution (minimum 100ul) into a micro-centrifuge tube and discard

the rest15. Now add 2ml of 10mM reduced glutathione solution into the column and let it drip out

(Collect 0.5ml fractions, a total of four micro-centrifuge tubes)16. Add 5ml of PBS to the column and let it drip out

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17. Discard the solution18. Add 2ml of guanidine hydrochloride solution to the column and let it drip 19. Discard the solution20. Add 5ml of PBS to the column and let it drip out into the centrifuge tube for 10min21. Discard the solution22. Add 2ml of 20% ethanol and let it drip out for 5min23. Close the bottom end of the column using the end cap24. Add 2ml of 20% ethanol to the column25. Close the column using the lid and give it back to TA

SDS-PAGE

Preparing protein sample:

Do the following for all the samples collected during purification

1. In a microcentrifuge tube, take 30 µl of bacterial cell lysate/flow through/purified samples and dilute it with 10 µl of SDS sample buffer to get 3:1 (v/v) dilution , vortex the samples.After adding SDS sample buffer follow the steps below and move the sample to water bath immediately. The longer you leave samples in SDS at room temperature the worst your gel will look

2. Close the tube cap – Make sure its tightly closed3. Now move the tube to the dry bath set to 95ºC and boil for 3 min4. Move the tube to an ice box till further use

Sample loading and running gel:

1. Take a gel cassette and lay it flat on the desk with the bottom tab with words break off facing towards you

2. Adjust the cassette as such that the bottom tab is sticking out of the table3. Now twist the bottom tab with your thumb and index finger to break it off4. Now gentle remove the well comb of the top of the cassette 5. Rinse the wells with 1X SDS buffer 6. Now move the cassette to a cassette holder and clamp it in tight,

Always maintain the cassettes and buffer chambers in upright position with samples wells facing up right. Sample wells should face inside the chamber such that when the upper tank is filled with buffer, wells also gets filled with tank buffer)

7. If running a single gel, use a dummy cassette to seal the other side of the holder

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8. Fill the cassette holder with small volume of 1X SDS tank buffer making sure that the sample wells are not filled with buffer

9. Check the holder for any leaks10. Now move the cassette holder to the SDS page electrophoresis chamber 11. Fill the lower part of the chamber with 1X SDS tank buffer until the bottom portion of

the gel is submerged.12. Now slowly fill the cassette holder which now acts as upper part of the chamber with 1X

SDS buffer so that the gel wells are completely covered with buffer13. Using a micro pipette tip (yellow tips) carefully add 10ul of prepared protein sample to

the bottom of the well14. TA will add protein ladder to be used as standard and positive control15. Connect the power supply to the chamber and run at 200V for 30-45 min16. After 30-45 min disconnect the power supply and remove the cassette holder

Disassembling gel and gel staining:

1. Transfer the buffer inside the holder and remove the cassette2. Now place the cassette on benchtop with some blotting paper3. Carefully break open the seals on the sides of the cassette carefully (TA will show you

how)4. Use the cassette half with gel on it to carry the gel to staining solution containing

coomassie blue.5. Shake the staining buffer container with gel in it for 2 min on an orbital shaker6. Carefully transfer the staining solution back into the bottle and wash the gel with

distilled water for 2 times.7. To the gel box add de-staining gel solution and keep it on rocker for 30 min.8. Gel is now ready for photo shoot.

Results

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Figure 1: Gel Reference for Molecular Weight

Figure 2: Results of the SDS-Page Gel Electrophoresis

Discussion and Conclusion

The results of the experiment indicate that complete isolation of the NcRFC protein was unsuccessful, however purification may have had partial success. The expected results are those of separate bands to indicate the different components of the protein sample, and of which have small differences in molecular weights for each band. By referencing the results of Figure 2 to the molecular weight reference of Figure 1, each sample group illustrated molecular weights of around 5-10 kDa. Each group also produced one large blob in this range as opposed to separate bands. The blob as opposed to band outcome could be due to problems in purification. These blobs indicate one collective substance as opposed to separate components, and therefore could’ve been unsuccessfully purified during affinity chromatography or isolated during SDS-Page. During either of these steps, the components could have, for instance, bound

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together or broken down completely to give one large component or infinitely many small components to explain the non-banded results. These results also could’ve been due to problems in the SDS-Page step that altered, for instance, the ability of gel electrophoresis to separate the molecular weights. If the purification process broke down the mixture into infinitely small components, then the SDS-Page readings prove accurate. However, if the mixture somehow bound together, then the gel pores could have trouble tracking the molecular weight of a bonded multi-component sample to give the results shown. However, the observance of Figure 1 and Figure 2 doesn’t completely prove unsuccessful. Due to the similar molecular weight ranges for each sample, it can be suggested that purification was performed to some degree. Components with much different molecular weights aren’t resulting, and therefore purification, most likely from affinity chromatography, was able to isolate to a very limited range of molecular weights. Therefore the SDS-Page results suggest purification was somewhat successful, but not completely, during affinity chromatography. In addition to SDS-Page, mass spectrometry could have been performed to further determine the purity of the samples. Mass spectrometry results would also provide whether the blob results were due to purification or problems with the SDS-Page. In conclusion, the objective was not met completely, however the results illustrate that part of the objective was met. While the SDS-Page results demonstrate that the NcRFK protein was not completely purified and isolated, they do demonstrate that a large degree of purification was met due to the small range of molecular weight outcomes.

Lab 2: Restriction Digestion

Jacob Feste

010617389

2/9/15

Objective

The objective of this experiment was to determine the impact that the restriction enzymes BamHI and XhoI have on a specific plasmid DNA. More specifically, this experiment was designed to determine the size of the plasmid DNA after the restriction enzyme cleavage for

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both BamHI and XhoI. In order to get a relative estimation of how far down the enzymes cleave the given plasmid DNA strands, each enzyme was tested using single digestion and double digestion, with a relative negative control for reference. They were also tested as a mixture of the two. The products of each cleavage trial as well as the negative control were subject to gel electrophoresis, with a 1Kb DNA ladder for size reference. Overall this experiment determines the effectiveness of each restriction enzyme for the given plasmid DNA.

Materials

BamHI/XhoI Single/Double Digestion

1. 12.5 micro liters plasmid DNA2. Three micro centrifuge tubes3. 1.5 micro liters of NEB Cut smart buffer4. 1.5 micro liters each of XhoI and BamHI5. Microcentrifuge6. Water bath

DNA gel electrophoresis

1. 320mg agarose2. Micro balance3. Weighing paper4. Screw cap glass bottle5. 0.8ml 50X TAE6. Water7. Microwave8. Casting mold and comb9. 10ml 1X TAE buffer10. Amount of 1X TAE to submerge gel11. 10 micro liters plasmid solution12. 2 micro liters 6X gel dye13. 1Kb DNA ladder14. Undigested pDNA15. 3 micro liters Ethidium bromide solution16. Gel scoop17. .05% Ethidium bromide bath18. Blotting paper19. Transilluminator (UV)20. Camera21. Power Supply

Procedures

BamHI/XhoI Single/Double Digestion

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1. 12.5 micro liters of plasmid DNA (pDNA) was taken into three micro centrifuge tubes and the tubes were labeled Xho1, BamH1, and Xho1/BamH1. The pDNA used was the DNA extracted from the miniprep procedure in the previous lab.

2. 1.5 micro liters of 1X NEB Cut smart buffer (pH 7.9) was added to the solution.3. The lids on the test tube sample(s) were closed and the samples were mixed gently by

gentle tapping of the tubes. The vortex mixer was not used.4. 1 micro liter of Xho1 and BamH1 were added to the respective test tubes, and for the

Xho1/BamH1 tube, 0.5 micro liters of each restriction enzyme was added. 5. Step 3 was repeated and the samples were spun in the microcentrifuge for 10 seconds.6. The samples were then incubated in a 37 degree Celsius water bath for one hour.

DNA gel electrophoresis

1. Begin by weighing out 320mg of agarose via a micro balance and weighing paper.2. Transfer the agarose to a screw cap glass bottle.3. Add 0.8ml of 50X TAE using a micro pipette.4. Next, add 40ml of water also using a micro pipette.5. The bottle cap is then lightly loosened and microwaved for 2 rounds of 30 seconds each.

Swirl the bottle gently after each cycle. The agarose should be completely dissolved after the second cycle.

6. Cool the solution by leaving the bottle under running water for 15-20 seconds. Don’t let the solution cool too much as to solidify in the bottle. Maintain a luke warm temperature.

7. Next, add 3 micro liters of Ethidium bromide solution to the bottle. Mix well. 8. Slowly add the solution to a casting mold with the comb in place.9. To generate gel formation, leave the solution in the mold for 15 minutes.10. Next, add 10ml of 1X TAE buffer to the newly formed gel.11. Holding the tube up straight, remove the comb slowly from the gel while making sure

not to bend the comb.12. The tank is then filled with 1X TAE until the gel is completely submerged.13. The Eppendorf tubes are then added with 2 micro liters of 6X gel dye.14. Mix the contents by pipetting up and down.15. Take 5 micro liters of this solution and carefully transfer it to the gel wall.16. Add 1Kb DNA ladder to the gel, as well as undigested pDNA.17. To perform the electrophoresis, begin by connected the gel chamber to a power supply.18. Using the power supply, apply 100V of electricity at 75 milli amps for 40 minutes.19. Remove the gel using a gel scoop after this time period after first turning off the power

supply.20. Then, transfer the gel into a .05% ethidium bromide bath inside of a chemical hood.21. Leave the gel in this solution for 15 minutes.22. The gel is then blotted in the chemical hood using blotting paper and transferred to a

transilluminator (UV).

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23. Turn on the transilluminator to illuminate the gel.24. Finally, use a camera to obtain photos of the result. Make sure to discard the gel into

biohazard waste.

Results

Figure 1: Gel electrophoresis results with the 1Kb DNA ladder, negative control, single digestion and double digestion tests

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Figure 2: 1Kb DNA ladder for comparison

Discussion and Conclusion

The results illustrated by figure 1 above indicate that the restriction enzymes BamHI and XhoI performed cleavage for both single and double digestion. This is indicated by referencing the experimental well results with both the negative control and 1Kb ladder wells. The 1Kb ladder, indicated by the 4th well from the left in figure 1, can be referenced as an increase in kb size the higher up the ladder, as illustrated by figure 2. The 5th well from the left indicates the negative control group, or untreated pDNA, and has the largest size at around 8-10kb. The BamHI results are illustred by the 6th and 8th well from the left, with the 6th indicating single digestion at around 6-8kb in length and the 8th indicating double digestion at around 4kb in length. The XhoI results are indicated by the 9th and 10th wells from the left, with the 9th well indicating single digestion at around 4kb in length and the 10th indicating double digestion at around 2kb in length. Finally, the mixture of BamHI/XhoI results are indicated by the last two wells from the left, with the second to last indicating single digestion and the last indicating double digestion. Both of these gave results of about 3-4kb in length. With the decreases in lengths for each of the tests, it can be concluded that both restriction enzymes performed cleavage on the pDNA. It can also be concluded that XhoI performed cleavage more often than BamHI due the smaller lengths of the resultant DNA. Double digestion seemed to create smaller DNA fragments for both the XhoI and BamHI enzymes than single digestion, with XhoI’s single digestion results about the length of BamHI’s double digestion results. However, the mixture of BamHI and XhoI results provided almost identical lengths for both single and double digestion. This is likely due to the each of the enzymes cleaving restriction side codons necessary for the other enzyme after the first digestion, causing the second digestion to be almost identical due to the lack of codons for further cleavage for each enzyme. These results were as predicted by the pre-lab. While it was difficult to predict the degree of cleavage for each enzyme due to the lack of knowledge of the pDNA’s specific codons, the degree at which these enzymes would cleave for single and double digestion could be foreshadowed. The negative control was expected to have the longest length due to lack of cleavage in the absence of a restriction enzyme. The single digestion tests were expected to shorten the length due to a round of cleavage, with the double digestion tests furthering that cleavage. Finally, the mixture was expected to give similar results for both single and double digestion due to vast altercations each enzyme could perform on its counterpart’s restriction codons after single digestion. In conclusion, the objectives were met as expected and each restriction enzyme performed its responsibility of cleavage on the subjected pDNA.