Proteomics

Isoelectric Focussing

Isoelectric Focussing

Proteins exhibit unique iso-electric property, such unique propertyof the proteins are exploited for the separation into individualproteins from a pool of proteome. Each single protein can beseparated from one another by iso-electric property.

Learning Objectives:

After interacting with this learning object, the learner will be able to:

• Operate steps involved in handling the instrument and thematerials 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.

• Define first dimension separation of proteins using Iso-Electric Focusing (IEF) system.

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.

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 itcompletely before use.

Cleaning and Drying the IPG Tray

Isoelectric Focussing

Place the instrument on leveled and shock freesurface. Now, position the manifold inside the grooveof the instrument. Ensure that the surface is even byplacing the round spirit level and making necessaryadjustment at the back edge of the instrument. Themanifold surface must be leveled, inorder to avoid anyleakage of cover fluid during the run.

Position the Manifold

Isoelectric Focussing

Soon after passive rehydration, pick the strip from thereswelling tray using forceps.

Drain out excess oil by tapping it on tissue paper byholding it straight not folding/bending it. Place thepositive side of strip on the positive end of theinstrument with gel side up.

Transfer IPG strips to Manifold

Isoelectric Focussing

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 1/3portion of it touches the gel surface.

The paper wicks absorb impurities /salts and aid incurrent conductance.

Moisten and place the Electrode Wicks

Isoelectric Focussing

Add cover fluid in all the wells containing the stripsand to the other wells. Maintain the level of coverfluid to prevent it from over flowing.

Pour Cover fluid

Isoelectric Focussing

Place the electrodes on either side of the tray suchthat the tip of the electrode touches the paper wickand is immersed in the cover fluid and close the lid.The electrode assembly must be placed between themarked places on the platform. Different markings areprovided according to the strip length which helps theuser to be sure that the assembly is placed correctly.

Position Electrode Assembly

Isoelectric Focussing

Connect the instrument properly. Switch ON thesystem and Start the software by clicking on the icon.Once instrument is ON it automatically calibrates tocheck all the parameters and working status. Once itis done, the system displays a ready status for the userto proceed.

Switch ON the instrument

Isoelectric Focussing

Select the specific protocol for the choice of thesample to run the IEF. The protocol must be specifiedand initial run must be performed before going for thefinal run.

Set program parameters and run IEF

Isoelectric Focussing

User can change and define the protocol according tosample requirements.

Set program parameters and run IEF

Isoelectric Focussing

User is provided with option to select the strip lengthand define the number of strips run for theexperiment.

Set program parameters and run IEF

Isoelectric Focussing

Once the protocol has been defined, it is connected tothe instrument and transferred to the instrument.

Set program parameters and run IEF

Isoelectric Focussing

Start the run. The set voltage is reached if the samplepreparation is perfect and the IEF run condition willbe achieved without any interference.

Set program parameters and run IEF

Isoelectric Focussing

However, the set voltage will not be attained if thesample contains impurities which may interfere duringthe run.

Set program parameters and run IEF

Isoelectric Focussing

If the running voltage deviates from set voltage, presspause icon to stop the focusing.

Set program parameters and run IEF

Isoelectric Focussing

In case of improper run replace the wicks with thenew one. The wick turning yellow is an indication ofabnormal run. The user needs to clean-up the sampleand perform the run if the wicks turn very darkyellow. Just in case the wicks are light yellow, thewicks can be changed and the focusing can be carriedout.

Set program parameters and run IEF

Isoelectric Focussing

Re-start the run by clicking on play button.

Set program parameters and run IEF

Isoelectric Focussing

The run continues once the wicks are changed.

Set program parameters and run IEF

Isoelectric Focussing

Under the influence of applied electric field, theproteins start moving till they reach a point where thenet charge on them is zero, this particular pointwhere the protein has a net charge zero is calledIsoelectric point. Each protein differs from the otherbased on the Isoelectric point and this property of theprotein is exploited during the separation of proteinsduring Isoelectric focusing.

Mechanism of Separation

Isoelectric Focussing

At iso-electric point the number of positive charges isequal to the number of negative charges present onthe protein.

Mechanism of Separation

Isoelectric Focussing

Stop the run once the set voltage is reached and therun is completed.

Completion of IEF

Isoelectric Focussing

Remove the IPG strip using forceps with care must betaken not to bend the strip and excess oil mustremoved by soaking over the tissue.

Storing the Strip

Isoelectric Focussing

Store the strip at -80°C until further processing. Oncethe equilibration buffer is prepared the strip can betaken out to carry out 2D run.

Do follow the future viewing IDD for moreinformation.

Storing the Strip

Isoelectric Focussing

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