Hi,
I am trying to represent my western blots for paper and have a clarification how to represent the Y axis in the graph. I normalized the test protein with actin and then divided my test protein with the control and kept the control value as 1. In this case how do I represent the Y axis in the graph. Do I represent it as fold difference or give as normalized to control. Inputs appreciated
Jaya
Hello Jaya,
If you have normalised with your housekeeping protein you can either represent the value 1 or just not do it if you clarify it enough in the figure legend. It depends on the purpose of the WB, if is just for the records, the more details the better to remember what you've done but if this goes for a publication it is better to research a bit on the journal or even better ask your supervisors what is the best way to do it. To me both ways are acceptable, you don't always have to include the 1 value bar to represent your control.
Hi Jaya
My opinion:
y axis title can be 'Values normalised by Untreated/wild type +/- SD'
Here your untreated value can be 1 or 100%.
Treatment/Untreated will directly give you comparison between untreated, treatment and control, if there is any.
Usually normalization by the loading control (actin) should be sufficient. here i am assuming you are calculating the band intensity using image J.
All the best.
My opinion can be wrong and will be glad to learn if I am wrong and what is more correct way to present wb bands in papers.
Kind regards
Omkar Panchal
Thank you for your answers. Omkar Iam using licor image scanning system to quantify the bands.
Hi Jaya,
I agree with the answers above. You can can either report the values of your normalisation (as actin normalised values) or make the control =1 (or 100) and report the fold of control (or % of control). Either way is fine.
Just a word of caution, I know that everyone use actin or other abundant protein as internal control and I can understand why. However when the protein is too abundant (and the antibody very good) the probability of getting a saturated signal is high. If this happens all your quantification is flawed. It seems you used the fluorescence secondaries and, I believe, the Liacor tells you when the signal is saturated, so I guess this is not your case... Of course this was a greater problem in the old days when the western were detected with ECL and films, but since I often noticed that this is an overlooked problem, I thought it was a good idea to mention in here.
good luck
Max
Thanks for your answer. Yesif the protein is satuarated, I cant quantify using licor system. Though it gives the picture, the values will go to infinity. So that is not the problem with my experiments
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