Wide Bands Trouble

[caleb]

Hi, I am having problems after runnning an electroforesis I get really wide bands, compared to the rest in the same gel.

To somewhat illustrate what happens, I load and run my gel and after doing the western we have to quantify the size of the bands to analyse the amount of protein loaded. Most bands come out to a size of 1mm (for example) and then there would be one or two that come out at 1.5mm. The DR says that this is not good for quantifying because they all should be the same or very similar in size.

Now, I know that I don't press too hard on the gel and I am following the protocol exactly, but it still happens. Is it that I am just still pressing too hard? I would appreaciate any help.

[kevin_a]

Since this is a Western, it's very likely that you are getting very high signal which is easy to fix by taking a shorter exposure, or loading less sample, or using using less antibody, or using a substrate that is not as sensitive.

High signal will cause saturation of film or CCD imager causing you get a plateau effect at high signal rather than the linear response you need for quantification.

[keithbrent2000]

Yes, you can try to run serial dilutions of your sample (half as much in the next lane as in the previous lane) to see how much sample gives the best signal.