This is the upper part of a western blot membrane, and as you can see there are some proteins that did not enter the separating gel.
Does this affect the level of protein expression? I mean is it wrong to take the lower band as a final result ?!
What could be the problem, and how to fix it?
Is it because I've loaded a hight amount of protein (60ug)?
Hi
Yes you loaded more protein. Some protein couldnt pass through your lanes, because it is aggregated. I think you overheated your sample or you forgot to spin your sample after heating. If you want to compare different lanes in this blot then t is not ideal to use this blot to quantify level of expression.
That blot is definitely overloaded. Even if you hadn't had aggregated (?) proteins left in the stacking gel, I'd be hesitant to quantify this gel. It might feel nice to see nice strong bands (intuitively we think high signals are good), but it also means we could be saturating the membrane. For determining the optimal amount of protein to load, I highly recommend the following paper: Article The Design of a Quantitative Western Blot Experiment
Lots of good tips on getting reliable, reproducible Western blots here. Good luck!
Loading 50 mcg protein could be the main reason.
I remember I had faced a similar problem. We found out that, the denaturation step (heating the sample with LB buffer containing SDS) also is a critical step. When you use the lamelli buffer, make sure that it is properly thawed and mixed well before its addition to the protein samples (SDS tends to settle down at the bottom if not mixed properly, and you would be trying to denature it without the most important component ie, SDS. This will not make the proteins separate).
It could also be because of improper sample preparation (like improper homogenization of the tissue).
So to summarize, do consider to look into the
1) amount of the loading protein
2) denaturation step
3) sample preparation step
To determine a good amount to load, you may want to first run a gel where you load different amounts of the same sample protein extract in 5 or 6 different lanes. I am guessing that for the highest amount, I would use one-fourth or one-fifth of what you have used in the above blot. For the lowest amount, i would use one-twentieth of the highest amount. write down meticulously every detail of blot development, including temperature and timing for each step. After scanning the blot, measure the intensities of the bands and plot against the amounts of protein loaded. The linear portion of the plot would define the range of sample loads that you would use for your actual experiment. if there is no linear plot, you may have to repeat with a different range of sample loads. For the actual experiment, try to adhere as closely as possible to the same blot development protocol.
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