Learning Objectives: Proteomics Isoelectric Focussing

Transcription

1 Proteomics Isoelectric Focussing Isoelectric Focussing Proteins exhibit unique iso-electric property, such unique property of the proteins are exploited for the separation into individual proteins from a pool of proteome. Each single protein can be separated from one another by iso-electric property. Learning Objectives: After interacting with this learning object, the learner will be able to: Define first dimension separation of proteins using Iso-Electric Focusing (IEF) system. Operate steps involved in handling the instrument and the materials used. Recall the background to Iso-Electric Focusing (IEF). Infer the steps involved to perform in the experiment. Assess the troubleshooting steps involved in the experiments. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.

2 Cleaning and Drying the IPG Tray Take out the tray/manifold and place it on the table. Clean all the lanes of tray/manifold with tissue paper, to make it free from dust, moisture and dry it completely before use.

3 Position the Manifold Place the instrument on leveled and shock free surface. Now, position the manifold inside the groove of the instrument. Ensure that the surface is even by placing the round spirit level and making necessary adjustment at the back edge of the instrument. The manifold surface must be leveled, inorder to avoid any leakage of cover fluid during the run.

4 Transfer IPG strips to Manifold Soon after passive rehydration, pick the strip from the reswelling tray using forceps. Drain out excess oil by tapping it on tissue paper by holding it straight not folding/bending it. Place the positive side of strip on the positive end of the instrument with gel side up.

5 Moisten and place the Electrode Wicks Cut the paper wicks into two pieces. Add distilled water to the wicks until it gets wet. Place the wick over the end of the strip such that 1/3 portion of it touches the gel surface. The paper wicks absorb impurities /salts and aid in current conductance.

6 Pour Cover fluid Add cover fluid in all the wells containing the strips and to the other wells. Maintain the level of cover fluid to prevent it from over flowing.

7 Position Electrode Assembly Place the electrodes on either side of the tray such that the tip of the electrode touches the paper wick and is immersed in the cover fluid and close the lid. The electrode assembly must be placed between the marked places on the platform. Different markings are provided according to the strip length which helps the user to be sure that the assembly is placed correctly.

8 Switch ON the instrument Connect the instrument properly. Switch ON the system and Start the software by clicking on the icon. Once instrument is ON it automatically calibrates to check all the parameters and working status. Once it is done, the system displays a ready status for the user to proceed.

9 Set program parameters and run IEF Select the specific protocol for the choice of the sample to run the IEF. The protocol must be specified and initial run must be performed before going for the final run.

10 Set program parameters and run IEF User can change and define the protocol according to sample requirements.

11 Set program parameters and run IEF User is provided with option to select the strip length and define the number of strips run for the experiment.

12 Set program parameters and run IEF Once the protocol has been defined, it is connected to the instrument and transferred to the instrument.

13 Set program parameters and run IEF Start the run. The set voltage is reached if the sample preparation is perfect and the IEF run condition will be achieved without any interference.

14 Set program parameters and run IEF However, the set voltage will not be attained if the sample contains impurities which may interfere during the run.

15 Set program parameters and run IEF If the running voltage deviates from set voltage, press pause icon to stop the focusing.

16 Set program parameters and run IEF In case of improper run replace the wicks with the new one. The wick turning yellow is an indication of abnormal run. The user needs to clean-up the sample and perform the run if the wicks turn very dark yellow. Just in case the wicks are light yellow, the wicks can be changed and the focusing can be carried out.

17 Set program parameters and run IEF Re-start the run by clicking on play button.

18 Set program parameters and run IEF The run continues once the wicks are changed.

19 Mechanism of Separation Under the influence of applied electric field, the proteins start moving till they reach a point where the net charge on them is zero, this particular point where the protein has a net charge zero is called Isoelectric point. Each protein differs from the other based on the Isoelectric point and this property of the protein is exploited during the separation of proteins during Isoelectric focusing.

20 Mechanism of Separation At iso-electric point the number of positive charges is equal to the number of negative charges present on the protein.

21 Completion of IEF Stop the run once the set voltage is reached and the run is completed.

22 Storing the Strip Remove the IPG strip using forceps with care must be taken not to bend the strip and excess oil must removed by soaking over the tissue.

23 Storing the Strip Store the strip at -80 C until further processing. Once the equilibration buffer is prepared the strip can be taken out to carry out 2D run. Do follow the future viewing IDD for more information.

24 Proteomics Extraction of Bacterial Protein

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