Quantifying Western Blot gels using BioRad Image Lab 6

This is a tutorial for using the program, Biorad Imagelab 6. Here we explain how to quantify bands relative to a standard protein amount.

[@revemystery5743]

what should be the standard?

[@luisp.9710]

Hi. By using the image lab software, can you select and work with 2 or more bands per lane. For exemple if my antibody recognizes 3 isoforms of a protein and detects 3 bands. ? Thanks

[@kellenjaxtyn3487]

a trick: watch movies on flixzone. I've been using them for watching loads of movies lately.

[@terrancejeffrey6175]

@Kellen Jaxtyn definitely, I've been using Flixzone} for years myself :D

[@yahyayozbatiran]

when I click the lane profile option, it shows me inverted peaks. How can I dissolve that problem?

[@underbubbles]

I've been playing with this and I'm almost certain that most people don't run a serial dilution of a given sample to validate quantity vs signal or band intensity (area under the curves) vs amount loaded. ie. What does a 20% increase or decrease or increase look like? I played with the exposure/maximum signal before saturation, and I get the best linearity of the antibody signal/quantity when the exposure is only 20-30% of the maximum.

[@HexIron]

Honestly this is good practice, but undoable in today's conditions.
A regular lab doesn't have neither the time nor the ability to keep up with all the controls.
I blame the mindset of fast publishing that pushes you to do quantity over quality.

[@revemystery5743]

where is your housekeeping gene? do we need it in quantification?

[@claraj6096]

I was wondering about that too, you definitely need it for quantification.. i think what she means with "standard" is just the positive control sample