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Proteomics

Gel Filtration Chromatography

Gel Filtration Chromatography

The method mostly involves the separation of the proteins basedon its molecular size. The proteins larger proteins are totallyexcluded from the gel and elute out first. The smaller proteinshowever, are small enough to move through the pores of the geland hence elute out after the larger proteins. This method is alsoknown as Size exclusion chromatography.

Learning Objectives:

After interacting with this learning object, the learner will be able to:• Prepare the column for the chromatographic technique.

• Prepare elution buffers for the experiments.

• Assess the troubleshooting steps involved in the experiments.• Analyse the mechanism behind the protein purification.

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.

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

Clean the surface of the balance and tare the weightof paper.

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

Prepare Elution buffer consisting of 0.1% SDS, 50mMTris dissolved in required volume of water. This bufferis used during equilibration and elution step. Whilepreparing the buffer, dissolve the componentsthoroughly using a magnetic stirrer.

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

Prepare elution buffer and adjust the pH to 8.5 usingNaOH. Just in case the pH goes higher than 8.5, bringit down using HCl.

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

Prepare size exclusion column using the beads withdefinite pore size which can separate protein based onthe molecular weight. Suspend the beads in theelution buffer and allow it to stand for 30 minutes.

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

The volume occupied by the beads should be 100times the sample to be loaded. Pour the elution buffercontaining the beads into the column and allow it tosettle.

Preparation Gel Filtration Elution Solution

Gel Filtration Chromatography

Equilibrate the column using Elution buffer.Equilibration ensures homogeneity in the beadsthereby bringing about proper separation of thesample.

Sample addition

Gel Filtration Chromatography

Load the sample to the gel filtration column forprotein separation.

Sample addition

Gel Filtration Chromatography

The separation is based on the molecular weight ofthe proteins in the sample, higher molecular weightproteins will be washed first while the proteins oflower molecular weight moves slower and takes timeto elute out as it passes through the pores of thecolumn.

Elution

Gel Filtration Chromatography

Pour the elution buffer to elute out the proteins.Elution buffer acts as mobile phase to elute theproteins from the column.

Elution

Gel Filtration Chromatography

The High molecular weight molecules tend to passthrough the openings between the beads, while thelow molecular weight molecules pass through the gelbeads taking a lot of time to elute out.

UV-visible spectrometry

Gel Filtration Chromatography

Detect the presence of protein using the UV-visiblespectrometry. The high absorbance reading indicatesthe presence of greater amounts of protein.

For more information on UV-visible spectrometry, gothrough IDD 50 on Basic instrumentation.