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Methodology for the labeling of proteins with cyanine dyes

Methodology for the labeling of proteins with cyanine dyes.

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Methodology for the labeling of proteins with cyanine dyes

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  1. Methodology for the labeling of proteins with cyanine dyes Cyanine labeling enables accurate analysis of differential expression of proteins between the samples. It is possible to label three different samples within the same 2-D gel, enabling accurate analysis of differences in protein abundance between samples by preventing gel to gel variation. • Related Los: Excitation and Emission properties, Protein properties > Prior Viewing – IDD-6. Extraction of serum protein, IDD-11. Protein quantification > Future Viewing – IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE • Course Name: Cyanine labeling • 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 1 After interacting with this learning object, the learner will be able to: • Define protein labeling techniques using dyes. • Select steps involved in handling the materials used. • Identify Mechanism of cyanine labeling. • Infer the steps involved to perform the experiment. • Assess the troubleshooting steps involved in the experiments. 2 3 4 5

  3. Master Layout 1 Preparation of working and stock solution of CyDyes (Slide: 5-7) 2 Sample preparation (Slide: 8-9) Cyanine Dye labeling (Slide: 10-12) 3 Stop reaction (Slide: 13) Store the sample at -20’C (Slide: 14) 4 5

  4. 1.Protein: Protein are the biomolecules composed of amino acid, forms the building blocks of the system and performs most of the biological function of the system. 2.Cyanine: is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging. Depending on the structure, they cover the spectrum from IR to UV. 3.Internal standards (reference standard): is a mixture of equal amounts of each sample, included in a each gel for an experiment. The use of internal helps for detection of spots and gives a great confidence to detect true biological variation. 4. Lysine: is a amino acid used to quench the labeling reaction. It acts as a substrate inhibition, binds to the excess dyes and helps to stop the labeling reaction. Definitions and Keywords 1 2 3 4 5

  5. Cy2 Description of the action/ interactivity Audio Narration (if any)‏ Instruct user to take out labeling kit from -20 C freezer. Let user opens the kit, show the tubes labeled with the CY3, Cy5, Cy2. the user should click on the tubes to get the structure of the dyes and with graph. Please redraw the figures Step 1: T1:Preparation of working and stock solution 1 2 3 Cyanine dyes are used for the differential labeling of the samples. Cy2 is yellow, cy3 is red and cy5 is blue in color. dyes have been designed to be both mass- and charge-matched. Spectrally resolvable with different excitation and emission wavelength. 4 5

  6. DMF Step 1: T1:Preparation of working and stock solution 1 2 3 Description of the action/ interactivity Audio Narration (if any)‏ Show the Dimethylformamide solution and the dye bottles, 3 fresh tubes(label as working stock). The user should click on the pipette, set to 5ul to draw the required volume of DMF add it to the dye tube and repeat the same for other two tubes , let user to do vortex (see next slide). Now from the solution, set the pipette to 5ul and take 5ul and transfer to fresh tube (working solution), do it for other two dyes. Now let user set the pipette to 7.5ul to draw DMF to add into working solution followed up with vortex. Transfer dye stock solution into its respective working solution. The step need to be carried out in dark room. Animate the step with user control. Prepare stock solution and working solution as per the desired concentration of the dye. DMF used to reconstitute the fluors should be high-quality anhydrous (>99.8% pure). It must not become contaminated with water, which will start to degrade the DMF to amine compounds. The DMF stock solution should be replaced at least every 3 months. 4 5

  7. Description of the action/ interactivity Audio Narration (if any)‏ Animate addition of DMF to dye tubes. Instruct user to carry out vortex each time. the user should keep the tube on rubber pad, click on the vortex to start the mixing of the solution. Step 1: T1:Preparation of working and stock solution 1 DMF 2 3 Vortex the content well. The final concentration of Dye has to be 400pmol/ul before carrying out the labeling step. 4 5

  8. Description of the action/ interactivity Audio Narration (if any)‏ Animate taking out stock samples (TEST and CONTROL) from the freezer by opening it and taking a box out and keeping it on ice-bath. Take a tube labeled as 25ug/ul control and other tube 25ug/ul test . User must click on the pipette to set to 50ul and Pipette 50ul of protein and add to tubes labeled as “control,test” Take out total of 25ul of TEST and CONTROL and pipette into Internal standard tube. Carry out the steps with user control with pipette action. Please redraw the figure. Step 2: T2:Sample preparation 1 STOCK Control TEST Internal Standard DMF 2 50ug 50ug 50ug EXPERIMENT Control TEST 3 Stock solution is sample solubilized in rehydration buffer after protein extraction. For labeling take protein sample equivalent to 50ug from test and control. For Internal standard for a single gel take mixture of test and control (1:1) equivalent to 50ug. 4 5

  9. Description of the action Audio Narration ‏ Step 2: 1 T2: Sample preparation 2 Colour of strip should be deep green-Alkaline pH 3 Animate like the user taking the pipette, setting the value to 5ul, pipetting out the sample from the tube and keep it on the paper (when the user clicks on it) as shown at one end now show gradually a change of color to dark blue. 4 The pH of the sample should be 8.8 for the accurate labeling and nest performance of the dye. 5

  10. TEST Internal Standard Control Description of the action/ interactivity Audio Narration (if any)‏ Step 3: 1 T3: Cyanine Dye labeling DMF 2 3 Show the working solution bottles labeled as Cy2, Cy3, Cy5. the user should click on the bottle so that the dyes are added to the respective tube. Instruct user to pick pipette, set it to 1ul, take out for say, cy2 and add in to internal standards tube, cy3 in test and cy5 in control. After addition change the color of the solution in the sample tube accordingly. Add 1ul each of cy2 to internal standard sample, cy3 to test samples and cy5 to control. There is no fast rule to use Cy3 only for control, Cy3 and Cy5 can be labeled either for control or test. But Cy2 must always be labeled to internal standard. 4 5

  11. TEST Internal Standard Control Description of the action/ interactivity Audio Narration (if any)‏ Step 3: 1 T3: Cyanine Dye labeling DMF 2 3 Show the mixing of the samples by vortex. The user should click on the vortex to start and later keep the samples on ice for 30 minutes in dark for labeling reaction to happen. Show a clock Mix the content well, vortex it and incubate in ice for 30 min in dark place. Avoid exposure to light, dyes are light sensitive. 4 5

  12. Description of the action/ interactivity Audio Narration (if any)‏ Step 3: 1 T3: Cyanine Dye labeling DMF 2 3 Zoom in the tube kept on ice bath to shoe the mechanism of labeling, animate a dye with NHS ester, going and binding to lysine group of protein. Positive charge on lysine protein must be replaced with dye. The cyanine dyes label the E-amino group of the lysine in the protein. when a CyDye is coupled to the lysine, replaces the lysine’s single positive charge with its own, ensuring that the pI of the protein does not change. Only 3% of total protein is labeled. 4 5

  13. TEST Internal Standard Control Description of the action/ interactivity Audio Narration (if any)‏ Step 4: 1 T4: Stop reaction Lysine DMF 2 3 Add 1ul of 10mmol/L of lysine to the samples and incubate for 10 minutes in ice. Free lysine help for quenching reaction, it goes and bind to free dyes. After 30min, instruct user to stop the reaction by adding lysine. Show the bottle labeled as lysine and the user should click on the pipette to set it for 1ul to draw lysine so that it can be added to the each tubes, followed by quick vortex and incubate on ice for 10min in dark. Animate the clock. 4 5

  14. Description of the action/ interactivity Audio Narration (if any)‏ Instruct user to store the tubes at -20’C into the freezer. Animate opening the door of freezer, placing the tube and closing the door. kindly redraw the images. Step 5: T5:Sample storage 1 2 3 • Now the labeled samples can be stored at -20’C or can be added into rehydration solution to make up volume required to load IPG strip to run IEF1D. Follow the future viewing IDD for more information 4 5

  15. Button 01 Button 02 Button 03 Slide 8-9 Slide 14 Slide 10-12 Slide 13 Slide1 - 4 4-7 Introduction Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Interactivity area Animation area • Provide user 20 samples(10 control and 10 test) to design DIGE experiment. • Instruction: animate to provide few options for user to select. Best option must be any 5 out of 10 control must be labeled with Cy3 and other 5 left must be labeled with Cy5. similarly even for test any 5 out of 10 must labeled with Cy5 and other 5 left must be labeled with Cy3. the internal standard must be pool of both test and control labeled with Cy2. • Provide user 20 samples(age from 25-35) to design DIGE experiment. • Instruction: animate to provide few options for user to select. Best option must be samples within the same age must be taken to run a dingle gel. For example sample1: 25.6years control and sample3: 25.8years test, these two must be grouped together to run a gel. Instructions/ Working area Credits

  16. Questionnaire: APPENDIX 1 Question 1 What is cyanine? • Glucose • Amino acid • Organic solvent • Organic dye Answer : organic dye Question 2 How does the cyanine label the protein? • By binding to the N-terminal of the protein • By binding to the C-terminal of the protein • By binding to the alpha amino group of the lysine • By binding to the Epsilon amino group of lysine Answer:By binding to the Epsilon amino group of lysine

  17. Questionnaire: APPENDIX 1 Question 3 What is the purpose of adding lysine? • To remove excess dyes • To react with protein • To stain the dye • To activate the dye Answer : To remove excess dyes Question 4 What is meant by internal standards? • BSA • Mixture of dye and sample • Mixture of test and control • Mixture BSA and test sample Answer: Mixture of test and control

  18. Questionnaire: APPENDIX 1 Question 5 Dye labeling changes the pI of a protein? • TRUE • FALSE Answer: False Question 6 The dyes are? a)Size and charge matched. b)only size c)only charge d)non of the above Answer: a) Size and charged matched

  19. APPENDIX 2 Links for further reading • Book: GE Healthcare handbook: 2D-Electrophoresis, principles and methods • Research paper: Tieqiao zhang et.al., Time-resolved excited state dynamics of cyanine dye, chemical physics letters: 2008, 236-240

  20. APPENDIX 3 Summary The samples and dyes has to be handled only in dark and on ice bath, as the dyes are light sensitive and temperature need to be maintained. The protocols has to be followed as per suggested to get appropriate result. The technology is based on the specific properties of spectrally resolvable dyes, that have been designed to be both mass- and charge-matched. Labeling of samples with three dyes, helps to run three different samples in a single gel.

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