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Data Analysis
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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