SDS-PAGE DATA ANALYSIS, create a standard curve (MUST MAKE A GRAPH ) USING EXCEL

Data Analysis

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Photograph the destained gel on an illuminated box each.

Create a standard curve of the protein standards plotting log of molecular weight

against distance moved from the wells.

Using a standard curve of the protein standards calculate the size of the GFP

purified in the:

o

Column purification samples

o

In the cell pellet lysates of the lb/ara/amp. You are only going to size the

band(s) that is darker in the lb/ara/ amp sample.

5

o

Prepare samples of each of the samples (tube 1,2,3 and lysate if you have it).

o

Heat all the samples at 95

o

C for 10mins

Cell Pellets

o

You will have two pellets from lab 9: ara and ara/amp

o

Add buffer to each tube as below

Components

Volume

Pellet

NUPAGE LDS Sample buffer

200ul

Total volume

200ul

o

Aliquot 20 ul of ara and ara/amp two separate tubes labeled ‘heat’

o

Aliquot 20 ul of ara and ara/amp two separate tubes labeled ‘no-heat’

o

Incubate the ‘heat’ samples for 10 minutes at 95

o

C

Sample Loading

o

Use the pipette equipped with a protein gel loading tip to underlay the samples into

the gel wells (see figure below).

o

Slowly draw the sample up into the tip as it will take time to fill due to the narrow

bore.

o

Lower the tip to the bottom of the sample well and slowly pipet sample into well

without contaminating another well with the sample.

Gel 1 – Samples for gel lanes

o

Protein ladder – 5ul

o

Tube #1 – 10ul

6

o

Tube #2 – 10 ul

o

Tube #3 – 10 ul

o

Lysate – 10 ul or leave blank

o

Protein ladder 5ul

o

Tube #1 – 15 ul

o

Tube#2 – 15ul

o

Tube #3 – 15 ul

o

Lysate – 15ul or leave blank

Gel 2 – samples lanes

o

Protein ladder – 5ul

o

amp – heat

o

amp- no heat

o

ara/amp- heat

o

ara/amp – no heat

o

protein ladder – 5ul

o

amp – heat

o

amp- no heat

o

ara/amp- heat

o

ara/amp – no heat

the first photo is Gel 1 , the second photo in Gel 2

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