1. Gel Filtration Chromatography The method mostly involves the separation of the proteins based on its molecular size. This method is also known as Size exclusion chromatography ‏  Related LOs: Column preparation, Chromatographic technique > Prior Viewing – IDD-6. Extraction of serum protein, IDD-42. Liquid chromatography - affinity chromatography > Future Viewing – IDD-38. Stable isotope labeling using amino acids in cell culture (SILAC), IDD-37. Isotope-coded affinity tags (ICAT),IDD-39. LC-MSMS data analysis  Course Name: Gel Filtration Chromatography  Level(UG/PG): UG  Author(s): Dinesh Raghu, Vinayak Pachapur  Mentor: Dr. Sanjeeva Srivastava *The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license

2. Learning objectives After interacting with this learning object, the learner will be able to: 1. Define the column preparation for the chromatographic technique 2. Prepare elution buffers for the experiments 3. Analyse the mechanism behind the protein purification 4. Assess the troubleshooting steps involved in the experiments 5 3 2 4 1

3. Master Layout 5 3 2 4 1 Column Preparation (Slide: 5-13) Sample addition (Slide: 14-16) Elution (Slide: 17-21) UV-visible spectrometry (Slide: 22-24)

4. Definitions and Keywords 5 3 2 4 1 1. Gel filtration chromatography: The protein separation is based on the molecular size of the protein, gel packing in the column and the molecular filtration efficiency of the gel 2. Size exclusion beads: The manufactured beads that has pore size depending on the molecular weight/size of the protein to be purified. These beads act as stationary phase 3. Elution buffer: Elution buffer consists of, 0.1% SDS, 50mM TRIS that can be used as the mobile phase to elute out the protein from the column

5. 5 2 1 4 3 Beaker Magnetic bead Audio Narration (if any) ‏ Description of the action Show magnetic stirrer instrument. Let user place the beaker on it. Display the beaker containing powder at bottom, liquid layer on top and a magnetic bead at the bottom. Instruct user to ON the instrument, let user control the speed nob and regulate it accordingly to control the mixing speed in the beaker. Animate powder getting into the solution. Show a turbid solution turning colorless Step 1: T1: Column Preparation Magnetic stirrer instrument helps for evenly distribution of solute into the solvents at faster rate. Magnetic stirrer

6. Step 1: Audio Narration (if any) ‏ Description of the action 5 2 1 4 3 Show a measuring balance, with display, ON, OFF and TARE/0 buttons on it. let user ON it, display reading as 0.000g, let user picks up the paper from the rack, makes 1/10 of folding on the sides and places it on the balance. Now the display reading changes to 0.003g. Instruct user to TARE the reading. And animate to click the tare button. Once user clicks it, reading must show ”0” When measuing with paper, the weight of the paper need to be tared from actual reading. Measuring balance T1: Column Preparation

7. Step 2: Audio Narration Description of the action 5 2 1 4 3 Let user pick up SDS, tris base, measuring cylinder from the rack and keeps it on the table next to balance. Instruct user to weigh 0.1g of SDS, let user tare the balance, user should click on the SDS bottle, uncap it, with help of spatula weigh the required amount on a paper over the balance. Display a gradual increase in reading with quantity addition. if the gram exceeds user should remove some quantity or if it less add the quantity to get the exact required amount. After weighing transfer the quantity to beaker. Now weigh 0.12g accordingly for tris base. SDS Tris Base Prepare Elution buffer consists of, 0.1% SDS, 50mM TRIS, which is used during the equilibration step. T1: Column Preparation

8. Step 3: Audio Narration Description of the action 5 2 1 4 3 Now instruct the user to take water bottle, open the cap, take 100ml measuring cylinder, measure 90 ml. Let user remove the excess water if level crosses 90ml mark. Transfer it to beaker. Now take the beaker, shake it to make a proper mix as shown in slide 10. Animate the powder getting into the solution. Now set the pH to 8.5 by using pH meter. Measuring cylinder helps in making up the final required volume. T1: Column Preparation

9. Step 4: Audio Narration Description of the action 5 2 1 4 3 Before the pH reading, pH instrument need to be calibrated with standards. Once with STD 1 at pH 7 and with STD 2 at pH 10. STD 1 STD 2 Display standard pH bottles and pH instrument and deionized water, discard bottle placed on a table. Instruct user to caliberate the instrument. Let user ON the instrument. Initially for the pH rod is dipped in in dipped in 3M KCl. Now show like user taking out the rod and washing it with deionized-water, let user cleans the rod with tissue. Now pick the STD 1, uncap it, dip the cleaned rod into the solution, user must click read button with display showing “7”. Now clean the rod and repeat the step to note down the reading for STD 2 and now the display should show “10” T1: Column Preparation

10. Step 5: Audio Narration Description of the action 5 2 1 4 3 Instruct user to set the pH for labeling buffer pH at 8.5. Now take the Elution buffer bottle, uncap it, dip the cleaned pH rod into the solution. User need to click on read button. Initially display must show a reading 6. now instruct user to add NaOH to adjust the pH. Now allow the user to click on NaOH bottle so that drops of NaOH should be added with filler, user need to mix the solution with glass rod, click on read button and the reading should anywhere near 11. let user keeps adding the NaOH drop till the pH display shows 8.5 and later transfer the beaker solution to 100ml measuring cylinder to makeup the volume to 100ml by clicking on water and adding it to that. All action should happen when the user clicks the hand image. Prepare elution buffer of pH 8.5. NaOH HCl T1: Column Preparation

11. Step 6: Audio Narration Description of the action 5 2 1 4 3 Let user pick up Gel filtration beads, measuring cylinder from the rack and keeps it on the table next to balance. Instruct user to weigh 25g of beads, let user tare the balance, user should click on the SDS bottle, uncap it, with help of spatula weigh the required amount on a paper over the balance. Display a gradual increase in reading with quantity addition. if the gram exceeds user should remove some quantity or if it is less add the quantity to get the exact required amount. After weighing transfer the quantity to beaker. Instruct the user to click on the bottle labeled as elution buffer and allow him to pour to the beaker with the weighed beads and allow it to stand for 30 minutes and animate a clock Gel filtration Beads Prepare size exclusion column using the beads with definite pore size which can separate protein based on the molecular weight. T1: Column Preparation

12. Step 7: Audio Narration Description of the action 5 2 1 4 3 Let user take out the empty column from the rack, fix the stopper to close. Animate like the user taking a beaker with beads and user click, should pour the beads into the column and once poured show the column as in figure. DEAE Column 1 Coulmn T1: Column Preparation The bead volume should be100 times than that of the sample to be loaded.

13. Step 8: Audio Narration Description of the action 5 2 1 4 3 Animate like the user tightening the knob at the bottom in the column. The user must click on the beaker labeled as elution buffer and animate like the user pouring the solution inside the tube. Now instruct the user to click on hands to place a beakers at the bottom of the columns and open the stopper. Animate like the liquid comes out of the tube in drop to the beaker and show like closing the stopper and show a liquid layer at the top of the column Buffer Equilibrate the column using elution buffer. stopper Column Buffer T1: Column Preparation

14. Step 9: Audio Narration Description of the action 5 2 1 4 3 Show a tube labeled as “sample” and the user should take the pipette set to 250ul, pipette out the sample and add to the column as shown in figure Events must happen as and when user clicks on the pipette, animate a clock for 10 minutes Load the sample to the gel column to carry out filtration process. stopper sample T2: Sample addition

15. Step 10: 5 2 1 4 3 Column 1 T2: Sample addition

16. Step 10: Audio Narration Description of the action 5 2 1 4 3 Animate like rings of different color with some small and large circles. the larger circle rings must move at faster rate while the small circle rings must move slowly by passing through the column. Please re-draw the previous figure. The separation is based on the molecular weight of the sample, higher molecular weight proteins will be washed first while the proteins of lower molecular weight moves slower and takes time to elute out as it passes through the pores of the column. T2: Sample addition

17. Step 11: 5 2 1 4 3 Elution buffer T3: Gel filtration Elution

18. Step 11: Audio Narration Description of the action 5 2 1 4 3 Pour the elution buffer to elute out the proteins at faster rate. Now instruct the user to take the pipette set 1000ul and take the elution buffer and add to the column show the increase in the volume in the column and the large/small circle movement as described in slide 20. Events must happen when the user clicks on it T3: Gel filtration Elution

19. Step 12 : 5 2 1 4 3 T3: Gel filtration Elution

20. Step 12: )‏)‏ 5 2 1 4 3 T3: Gel filtration Elution

21. Step 13: Description of the action 5 2 1 4 3 Show the collection tubes in a row and the solution dropping into it. Show the tube:1 with only solution, tube:2 with some large rings, user should click on it and a tab should appear labeled as “high molecular weight proteins” and tube:3 with more of small rings tube:4 with some small rings user should click on it and a tab should appear labeled as ” Low molecular weight proteins” and decreased amount of small rings in tube:5,6 and less of small size rings in tube:7,8 and only solution in tube:9 T3: Gel filtration Elution The larger sized molecules tend to pass through the openings between the beads, while the small molecules pass through the gel beads taking a lot of time to elute out. Audio Narration

22. Step 14: 5 2 1 4 3 T4: UV-visible spectrometry Cuvette Please re-draw the figure to show Blue and green beads of different sizes

23. Step 14: Audio Narration Description of the action 5 2 1 4 3 Show a instrument labeled as “UV –visible spectrometry” and the samples in the stand as shown in figure. Animate buttons like “start, auto zero, absorbance, stop” on the instrument Now instruct the user to switch on the instrument, set the wavelength to 595nm by pressing on numbers-open the lid of the instrument and take a cuvette as in figure and click on phosphate buffer to take it into cuvette and animate like keeping it inside the UV Visible spectrometry and press “auto zero” Display a value on the system as “0.000” animate like the user opening the lid and taking out the cuvette and discarding the solution, now animate like the user taking the sample 1 and adding to the cuvette, keeping it inside, closing the lid and press absorbance show the values as in next slide for each sample follow the same for (2- 9) Detect the presence of protein of using the UV – visible spectrometry. The high absorbance reading indicate the presence of protein. For more information on the UV- Visible spectrometry please go through IDD-50 basic instrumentation. T4: UV-visible spectrometry

24. Step 14: 5 2 1 4 3 T4: UV-visible spectrometry Sampleabsorbance 10.12 20.229 30.303 40.457 50.533 60.681 70.71 80.62 90.65 Volume of sample 2 2 2 2 2 2 2 2 2

25. Animation area Instructions/ Working area Credits Name of the section/stage Interactivity area Tab 02Tab 03Tab 04Tab 05Tab 06Tab 07 Button 01 Button 02 Button 03 Tab 01 Slide 5- 13 Slide 14-16 Slide 17-21 Slide 22-24 Slide 13 Let the user remove the buffer entirely from the column, show like the column drying. Instruction Instruct the user to add the buffer to the column and close the stopper, show like some buffer still remains in the top of the column.

26. Questionnaire: APPENDIX 1 Question 1 In gel filtration chromatography separation is based on a)Molecular size b)Molecular structure c)Confirmation d)Stereochemistry Question 2 Gel filtration chromatography also known as a)Size exclusion chromatography b)Ion exchange chromatography c)Affinity chromatography d)Chromatofocussing Question 3: In gel Filtration chromatography mobile phase is a)Buffer containing acid b)Buffer containing Base c)Buffer Containing salt d)Size exclusion beads

27. Questionnaire: APPENDIX 1 Question 4: In gel Filtration chromatography stationary phase is a)Buffer containing acid b)Buffer containing Base c)Buffer Containing salt d)Size exclusion beads Question 5: Volume of sample to be loaded in the column is a)Sample volume=100*column size b)Column size=100*sample volume c)Samplevolume=column size/100 d)Sample volume= column size

28. Links for further reading Reference websites: 1. http://www.mnstate.edu/provost/sizeexclusionprotocol.pdf 2. http://kirschner.med.harvard.edu/files/protocols/GE_gelfiltr ation.pdf APPENDIX 2

29. Summary APPENDIX 3 Size exclusion chromatography involves separation based on molecular size of the protein. The size of the gel beads used in the column preparation plays a very important role in separation of molecules.