1. Affinity Chromatography Affinity chromatography is based on the principle of specific interaction between the protein or antigen and antibody for separation of biomolecules  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: Affinity Chromatography  Level(UG/PG): PG  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 required for experiment 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 Sample loading Sample separation UV-visible spectrometry Slide 5-6 Slide 9-14 Slide 15-17 Slide 7-8

4. Definitions and Keywords 5 3 2 4 1 1. Affinity purification: A chromatographic purification procedure that makes use of specific interactions between the analyte of interest and the capture analyte immobilized on the column. In some application Ni-His, iron,zinc, gallium column are used for analysis and in ICAT, avidin affinity chromatography is employed due to its specificity of interaction with biotin. 2. Isotope Coded Affinity Tagging (ICAT): ICAT is an in vitro labeling technique that modifies peptides or proteins specifically at the cysteine amino acid residue and can be used for accurate quantitation of protein expression. 3. Light ICAT label: The light ICAT reagent consists of a Cys-reactive group, an ICAT linker consisting of hydrogen atoms and a biotin tag. The chemically reactive group forms covalent bonds with peptides or proteins while the affinity tag enables the protein to be isolated by affinity chromatography in a single step that is used to tag the control sample. 4. Heavy ICAT label: The heavy ICAT label consists of a Cys-reactive group, an ICAT linker consisting of heavy deuterium isotope and a biotin tag that is used to tag the Drug treated sample.

5. Step 1: Audio Narration Description of the action 5 2 1 4 3 Instruct the user to go through the IDD-37. Isotope-coded affinity tags (ICAT) slide from 31 to 47. DEAE AVIDIN-Column Coulmn The samples for analysis is labeled with biotin tag. T1: Column Preparation

6. Step 2: Audio Narration Description of the action 5 2 1 4 3 Animate like the user taking a column from the fridge, by opening it and removing the cover and tightening the stopper at the bottom in the column. Zoom in to show the column as above DEAE AVIDIN-Column Coulmn Take the avidin column from the fridge to carry out pre-treatment of the column. T1: Column Preparation stopper

7. Step 3: Audio Narration Description of the action 5 2 1 4 3 Animate like the user tightening the stopper at the bottom in the column. The user must click on the beaker labeled as “distilled water” and animate like the user pouring the water inside the tube using the pipette by setting full volume. Now instruct the user to place a beaker at the bottom of the columns and open the stopper. Animate like the liquid coming out of the tube like drops into the beaker and show like closing the stopper. Distilled water Wash the column using distilled water to remove the buffer material used in column storage. stopper Column water T2:Sample loading

8. Step 4 Audio Narration Description of the action 5 2 1 4 3 Show a tube labeled as “tube 5,6” from slide: 56-57 of IDD-37. Isotope-coded affinity tags (ICAT) and user should take the pipette set to 100ul, pipette out the sample and add to the column as shown in figure. Repeat the step until all the samples are in the column. Events must happen as and when the user clicks on the pipette animate a clock for 10 minutes Load the sample to the avidin column. stopper sample T2:Sample loading

9. Step 5: 5 2 1 4 3 Column 1 Avidin labeled beads T3:Sample separation

10. Step 5: Audio Narration Description of the action 5 2 1 4 3 Animate like rings of different color labeled as Light (L) and similarly other rings of different colors labeled as Heavy (H) show like the L and H binding to the column while rings without H or L labeling comes out. Please re-draw the above figure. The separation is based on the affinity of the biotin in the tag to the avidin in the column, the protein which have taken up heavy and light isotopes tend to show affinity to the column. T3:Sample separation Light (L) Heavy (H)

11. Step 6: Audio Narration Description of the action 5 2 1 4 3 Pour 0.1% formic acid containing 0.1ml formic acid and 99.9 ml water to the column, to elute the bound molecules. Now instruct the user to take the pipette set 1000ul and take bottle labeled as 0.1% formic acid to add into the column. show the increase in the volume in the column and the circles with different labeling moving. show movement as described in slide:9 and 10. Events must happen when the user clicks on it T3:Sample separation

12. Step 6: 5 2 1 4 3 T3:Sample separation

13. Step 6: )‏)‏ 5 2 1 4 3 T3:Sample separation

14. Step 7: Description of the action 5 2 1 4 3 Show the collection tubes in row and the solution dropping into it. Show in tube:1 only the solution, tube:2 with some unlabeled rings, user should click on it and a tab should appear to display ”unlabeled proteins” and tube:3 with more of unlabeled rings, tube:4 with some labeled rings user should click on it and a tab should appear labeled as ”Isotope tagged proteins” and increased amount of L and H rings in tube 5,6 and less of L and H rings in tube 7,8 and only solution in tube 9. Instruct the user to take the tube 5,6,7,8 for analysis The protein that show low interaction with the beads will be eluted first followed by moderately interacting protein and highly interacting proteins. Here the proteins that are tagged with biotin label show more interaction when compared to other proteins. T3:Sample separation Audio Narration

15. Step 7: 5 2 1 4 3 Cuvette T4: UV-visible spectrometry

16. Step 7: 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, take a cuvette as in figure and click on phosphate buffer to take it into the cuvette and animate like keeping it inside the UV Visible spectrometry. Press “auto zero” display a value on the system as “0.000”. animate like the user opening the lid and taking the cuvette and discarding the solution, now animate like the user taking the sample:1 transferring it to the cuvette, keeping it inside, closing the lid and press absorbance to show the values as in next slide. For other sample (2-9) follow the same step like above. 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

17. Step 7: 5 2 1 4 3 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 T4: UV-visible spectrometry

18. Animation area Interaction 1: slide-16: user getting zero reading from tubes 6-9. Instructions: user need to increase the concentration of elution buffer to remove out the bound proteins. 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- 6 Slide 9- 14 Slide 15-17 Slide 7- 8

19. Questionnaire: APPENDIX 1 Question 1 In affinity chromatography separation is based on a)Molecular size b)Molecular structure c)specificity d)Stereochemistry Question 2 Affinity chromatography mostly uses a)Antibody coated beads b)Column with empty beads c)Only mobile phase d)Only column Question 3: Avidin-Biotin chromatography mobile phase is a)0.1% formic acid b)Buffer containing Base c)Buffer Containing salt d)Size exclusion beads

20. Questionnaire: APPENDIX 1 Question 4: In affinity chromatography stationary phase is a)Buffer containing acid b)Buffer containing Base c)Buffer Containing salt d)Beads with specific proteins/metal that react with tagged protein Question 5: biotin binds a)Antibodies b)Streptavidin c)Antigen d)column

21. Links for further reading Reference websites: 1. www.mnstate.edu/provost/sizeexclusionprotocol.pdf 2. www.younglin.com/brochure_pdf/waters/lcGPC.pdf APPENDIX 2

22. Summary APPENDIX 3 Affinity chromatography involves separation based specific interaction between the tagged protein and packing column used. The packing material used for the separation plays a very important role in separation of proteins. Steps involves: elution buffer preparation, sample loading, elution and analysis using UV-Visible spectrometry.