Passive and Active Rehydration - IIT Bombayoscar.iitb.ac.in/onsiteDocumentsDirectory/Passive... · Passive and Active Rehydration In active rehydration, sample is loaded in a single

Proteomics

Passive and Active Rehydration


Purified proteins in the rehydration buffer has to be loaded intothe strips, such that the strips take up the proteins into the gel forprotein separation during IEF. The process can be carried out inpresence and absence of current.

Learning Objectives:

After interacting with this learning object, the learner will beable to:• Identify steps involved in passive and active rehydration.• Determine the importance of passive and active rehydration.• Infer the steps involved in performing the experiment.• Assess the troubleshooting steps involved in the experiment.

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 manifold and place it on the table. Cleanall the lanes of tray/manifold with tissue paper, tomake it free from dust, moisture and dry itcompletely before use.

Cleaning and Drying


Fold a fresh butter paper and tare its weight from thebalance.

Preparation of Rehydration Solution


Prepare rehydration Solution consisting of 8M urea,2%CHAPS, 40mM Dithiothreitol and add 1%bromophenol blue followed by distilled water. Thissolution helps in protein solubilization. The volumerequired can be calculated by the number of the stripsused for the run accordingly.



Dissolve all the reagents in water firstly and later addthe required quantity of bromophenol blue(BPB).



Prepare the samples for loading onto IPG strips. About600ug of protein can be loaded for an 18cm strip. Ifuser is loading for 7cms and 24cms, then proteinamount can be calculated accordingly depending uponsample availability. To the sample add IPG buffercorresponding to the pH strip used and make up thefinal volume by adding rehydration solution. Give ashort spin to the tube to ready it for loading onto thestrip.

Sample Addition


Load the sample in the manifold in one shot, avoid airbubbles. The distribution of the sample should beequal and uniform all along the length of the strip.

Sample Loading


Remove the strip from -20°C and allow it to thaw for 5minutes, just before placing it over the samplesolution.

Overlay of Strips


Place the IPG strip in contact with the sample facingthe gel side down and keep it for half an hour.

Overlay of Strips


Add cover fluid to the strip, and avoid air bubbleswhile addition. Ensure that the strip is covered withoil totally. This prevents evaporation and helps toattain the set voltage across all the wells.

Application of Cover Fluid


Overnight process helps the IPG strip to getrehydrated with protein sample solution in absence ofelectric field. Once the passive rehydration is over,the strip is ready for IEF run. Please refer the IDD ofIEF.

Passive Rehydration


In active rehydration, sample is loaded in a singlemanifold electrode unit in case of single strip run orstrips loaded in the 12-well manifold. Sample layinginto the well and placing of IPG strip is the same likepassive rehydration. Unlike using of rehydration traylike in passive rehydration, for active rehydrationmanifold is used. The cover strip oil laid on the strip issaved.

Active Rehydration


The electrode assembly are held together and placedat the correct position by the plastic support. Thesystem goes in for automatic calibration for display,buzzer and light to check for working condition.

Active Rehydration


In active process, the condition for sample rehydrationis around 5hrs for 50-100V depending upon the sampleand it continues with the IEF run. Refer to the futureIDD for more information.

Active Rehydration