Hi Anna,

I met with the same problems as you some time ago, so I send you several sources regarding normalization in Western blot, have a look, maybe you will find some interesting idea...

Good luck

YOUR TAKING SAME NUMBER OF CELLS I MEAN EACH TIME WHILE REPEATING UR USING SAME NUMBER EXAMPLE ,,,, 1LACK CELLS PER ML ..LIKE ALL THE TIME IN ALL CONDITIONS....

Hi, Anna, do you mean biological or techinical repetitions? there are many factors which may have effect on absolutely value. BUT if yiou have same trends, you can consider it as true. From biological repetition, of course!

Hi Anna,

You need to consider an internal control sample and you need to run the sample any time you are running the gel for biological replicates, therefore you would be able to normalize your data against the internal control. It may help to reduce huge error bars! 

Hmm I have internal control (loading control), in the same gel... Differences are between biological replicates, but sometimes also between technical ones... I have loading control in the same gels and untreated control on the same gel, I load always the same amount of total protein... so the only think which could really affect my blots is transfer, I guess...

Trends are the same, but I am not sure how to present my data with so huge errors bars.. Ok, I will try perform more trials...

Thanks!

I would think just one possibility. The loading control you use are affected by the treatment? Normally, actin and GAPDH are OK but there is a possibility that the drug you use affects loading protein. I would suggest to use several different loading controls. 

Hi Anna, your problem may be related to protein estimation, or loading, or loading control. Try with another loading control. See that there is no saturation of bands.

Hi Anna,

As written above already, you can get variability from several issues. First, some general issues you have probably thought about already :

- your biological samples : if you work on cell lines for instance, avoid using one day freshly thawed cells and the other day high-passage cells.

- your compounds : do you use fresh aliquots each time ? Could it be a little more degraded each time you take it from a freezer ?

But if it is really WB-specific, I would bet on this issue : since you have a loading control, you use 2 antibodies on each experiment, let's say it is Gizmo vs Actin. First, if you recycle the antibodies, one can degrade faster than another, of course. But that's not over. If Gizmo signal is little vs Actin signal, you may saturate (even partly) Actin signal to see Gizmo by overexposure, and if saturation is not equal in every experiment (it depend on the variability of efficiency of your incubations), you get big errors. Avoid saturation by assessing it, do 2 different revelation steps for 2 antibodies (this is a basics, ok).  But also : even without saturation, if antibodies incubations efficiencies are not exactly the same between experiments, sometime Actin signal will vary between WB while Gizmo signal is stable while sometimes it will be the contrary. At the end : big variations anyway, even with consistent tendencies.

The solution to my mind : achieve robot-precision, or do more experiment to lower variations, use S.E.M. instead of S.E. if you have run a too little number of WB (I think it is ok for a few experiments), or plot the tendency instead of absolute values if possible (e.g. : between "minus compound" and "plus compound"  I have an average decrease of 50% of Gizmo, even if sometime it is 4 to 2 and sometime 40 to 20), or else show a "representative experiment" if this result is not too central in your paper. i hope it helps.

Thank you all a lot! I will keep in mind your advice!

Hi Anna,

Thinking about your question, the trend is the same based on a control band for that particular gel run, hope I am right here. The quantification was done and normalized for that control protein to say the trend is the same for your protein of interest. If both of them are correct, then the ratio of your protein to control should not be varying to give a huge error bar. You could represent this data as ratio to the control with proper error bars.

Good luck.

Hi Anna,

I met with the same problems as you some time ago, so I send you several sources regarding normalization in Western blot, have a look, maybe you will find some interesting idea...

Good luck

Are you saturating the film? Do you have access to an imaging system that would acquire the signal (ECL, presumably) directly from the membrane without the need for film exposure and development? That alone may reduce the variability.

What are you using to measure the intensities?

If the trend is the same, can you report your results in percentages or fold concentration change?

One other thing. You can cross check your WB results by doing dot blot. If the latter is also giving no significant results, then the WB results are correct!