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titleSetup

Well 1

HEK293

Well 2

B-Cells

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

GFP 500 ng

Dummy DNA 500 ng

Well 5

EMPTY

Well 6

EMPTY

Well 7

HEK293

GFP 500 ng

Syk-TEVp 500 ng

0X 0 nM Dox

Well 8

HEK293

GFP 500 ng

Syk-TEVp 500 ng

1X nM Dox

Well 9

HEK293

GFP 500 ng

Syk-TEVp 500 ng

10X nM Dox

Well 10

HEK293

GFP 500 ng

Syk-TEVp 500 ng

100X nM Dox

Well 11

HEK293

GFP 500 ng

Syk-TEVp 500 ng

1000X nM Dox

Well 12

HEK293

GFP 500 ng

Syk-TEVp 500 ng

2000X nM Dox

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titleExpected Results

Since Syk-TEVp should run at a different band size than endogenous Syk, we should be able to identify the presence of both or either on a Western blot using anti-Syk antibodies. (There should be two bands, one corresponding to Syk-TEVp and the other to endogenous Syk.) We can use GAPDH (which we assume is produced at the same level in all of the cell populations we are testing) to normalize our data. This way, we can make comparisons between different cell populations while controlling for the amount of protein that we loaded in each lane. After washing the paper blot with anti-Syk and anti-GADPH, we should get three bands based on the sizes of the proteins:

ProteinSize (kDa)
Syk72.08
Syk-(15aa)-TEVp100.5
GAPDH37
GFP27

GFP is added to control for variations in transfection efficiency for each transfection in the dox ladder. From the intensity of these bands, we should be able to determine the relative levels of endogenous and exogenous Syk expression.

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