1. Explain the principle of column chromatography. 2....

1. Explain the principle of column chromatography. 2. Consider 4 proteins with the properties shown in the following table. Answer the questions below about these proteins and justify your answers. Protein | Subunit Mass (Da) | Native Mass (Da) | pI | Binds S-hexylglutathione 1 | 5,000 | 20,000 | 8.1 | no 2 | 25,000 | 50,000 | 4.3 | no 3 | 10,000 | 10,000 | 7.8 | yes 4 | 12,500 | 37,500 | 3.3 | yes a. Suppose you want to isolate protein 1 from proteins 2, 3 and 4. List the order of methods you would use to purify protein 1. For each method you list, explain why you selected the method (in other words, explain which protein(s) are retained and which are eliminated). b. Diagram the SDS-PAGE electrophoretogram output that you would see as you monitored the purification conducted in part a.

Transcript

00:01 Hi there in this question different questions are asked from chromatography topic.

00:07 So, the first one first question is asking us column chromatography.

00:15 So, any type of chromatography there are two phases one is the solid phase sorry mobile phase and another is the stationary phase.

00:24 Okay so in case of column chromatography the principle is adsorption okay the principle is adsorption here a mixture of component okay which is known as the mobile phase okay it is dissolved in the column and it adsorbs into the column okay it is not absorption rather adsorption that means it remains adsorbed on the surface it is not absorbed into the column.

01:01 So, according to their affinity the mixture components the original components which were mixed supposed one and two were mixed in the mobile phase one and two will be then separated according to their affinity with the stationary phase.

01:19 This is how column chromatography works and the next question it is asking us about a different type of processes or chromatography techniques which we can use for isolating protein one.

01:37 So, first thing we need to do is the size exclusion chromatography size exclusion chromatography for chromatography word i'm not writing again and again it is c dot c dot means chromatography here.

01:56 So, in sec or size exclusion chromatography here according to their relative mass the components or proteins can be separated.

02:10 So, here protein one which is approximately 20 ,000 dalton will be separated from protein two okay which has a 50 ,000 dalton and protein three which okay so protein one protein two protein three and protein four.

02:30 So, they have different sizes okay dalton is given protein four has 37 ,500 dalton.

02:39 So, they will be partially separated first according to their relative mass okay.

02:50 Then next one we can use next thing we can use ion exchange chromatography and this is process number two iex okay or iec...

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