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BE115 1

Image J Protocol

For Western blot analysis

2/10/2006

Terry D. Johnson


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BE115 2

1. Open Image J an! in it! open the .TIF "ile to be

analy#e $ in this case! %e&ample.ti"'.


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BE115 3

1b. Optional

( I" the image is not alreay an )$bit .TIF! go to

Image -> Typean select 8-bit.

( I" the image is not ar* eno+gh to analy#eproperly! go to Image -> Adjust ->

Brightness/Contrastan a,+st the slier

bars +ntil the bans on the image are -isible.

The pi&el -al+es ill not be change! ,+st the-ie.


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2. In the main men+! select Analyze Calibrate.


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BE115 5

. elect the Uncalibrated ! option "rom the

"unction# ropon bo&! then clic* .


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BE115 6

3. 4 logarithmic plot sho+l appear5 mo-e it to the sie!

b+t o not close the ino.


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BE115 7

. 7o bac* to yo+r .TIF image an select the $ectangle

Tool "rom yo+r toolbo&. 8o& all o" the bans o" interest

across all o" the lanes an hit Crtl-% to bring +p a

histogram.


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BE115 8

6. 9are"+lly o+tline the "irst lane ith the same tool. 7o

to Analyze &els 'elect "irst (ane.


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BE115 9

:. Drag the rectangle that yo+ +se "or the "irst lane

o-er to the ne&t! an mar* it ith Analyze &els

)ar* +e,t (ane Don;t ra a ne rectangle< =o+ ant

e-ery rectangle to ha-e the same area. >epeat +ntil allthe lanes ha-e been mar*e.


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BE115 10

). 9lic* on Analyze &els .lot (anes. =o+;ll en +p

ith an image "ile ith plots "or each o" the lanes that

yo+ mar*e. The plot on top is the "irst lane that yo+

mar*e. ?se Image $otate 0 !egrees $ight 1or(e2t3 an/or Image 4oom ut so yo+ can see an

or* ith all o" the plots.


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BE115 11

@OPTIOA4BC I" yo+ ha-e more than one ban per lane!

yo+;ll en +p ith a plot "or a single lane that has

m+ltiple pea*s @one "or each banC.

=o+;ll nee to separate them into ini-i+al pea*s ith

the (ine Toolas emonstrate belo on another

image


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BE115 12

. elect the )agic 5and tool an select each plot

beginning ith the "irst. 4 bo& ill appear ith the 4rea

meas+rement "or each plot as it is selecte. The area o"

that histogram plot represents the ar*ness o" eachban in the gel.


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BE115 13

10. Once yo+ ha-e selecte each ban! yo+ can

analy#e the area meas+rements in yo+r spreasheet o"

choice.

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