Quantifying Western Blot gels using BioRad Image Lab 6

[@revemystery5743]

what should be the standard?

[@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.

[@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

[@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

[@DL-go7xj]

This video was not helpful for someone who has never imaged a western with image J. You skipped over steps and why you did the things you did. You spent a lot of time adjusting the background subtraction and didn't say anything about WHY. You neglected to say that you need an extra lane for the ladder. Even the type of comments show just how much you did not explain.