determining protein expression level from a Western!!! - (Aug/29/2005 )

i want to quantify the expression level of my protein in different cell lines. as a reference i used actin and the program im using is quantity one from biorad.

i have several doubts how to do it correctly:

1. should i draw a box of the same size for all the bands or a diiferent size depending on band´s thickness?

2. which value then is it better to take? the mean intensity in the box i drew? the intensity times area (if the boxes are of different size)? the values of all the pixels in the area? or shall i set the strongest ban to 100% and compare the rest to it?

im trying to figure this things out and i see that it´s difficult to avoid a biased result so i will be very greatful for adivce!!

cheers

cmon guys, none of you knows?

please...

I don't know how to answer this question, but I think I saw this same issue talked about before on this forum. Take a look around, you might find it here somewhere!
monkeybaji

i read all pf the discussions about this topic and but it seems that there are very different opinions abot that!

And thats just how it is, everyones has a different method for analyzing data. For imageJ I draw one big box around the scanned bands and plot the lanes using that. THen just draw lines that demarcate where the spaces are and then use the wand to measure the space. Confused? so am i, but it works reliably and interchangably with other methods of analysis

thank you bosco. im collecting opinions on different methods so i can figure out which one is the best for me.

how exactly do you separate the areas of the bands in the plot.? i try to draw a straight line but it disapears when i use the wand