I'm unsure exactly of what you are trying to ask, typically analysis is fairly straightforward, as you have a band (or no band, as the case may be) at a specific molecular weight. First, I would say be sure you are marking where your ladder markers are on your film after developing before moving your membrane. I use a piece of lab tape to secure my membrane in the exposure cassette so that I know it hasn't moved since I placed the film over it. I also make sure to push the film into one of the corners of the exposure cassette so that, after developing, I can replace it exactly where it was when I exposed so I can label where the bands were in the ladder.
If you want to quantitatively compare protein levels between samples, you need to also blot for another protein that should be equal in all of your samples (typically for eukaryotes a cytoskeletal protein is used for this such as actin or tubulin). Then you can determine the ratio of your protein vs. the control protein in the same lane. This can ONLY provide you with ratiometric information, however (e.g. there is more protein in lane 2 than lane 3), not actual quantities of the protein in terms of molecule number or concentration. This is because every antibody behaves differently and the affinity of the antibody against your protein is very unlikely to be the same affinity as the antibody used against your control. Even if you somehow knew these affinities were the same, you would need to know exactly the amount of control protein that was in your samples to determine the amount of your protein of interest.
With all of that said, there is a section in Bio-Rad's information manual on western blotting about "Data Analysis." Check it out here (starts on page 29): https://www.bio-rad-antibodies.com/static/Lit-pdfs/Brochures1/western-blotting.pdf
The Human Protein Atlas has set forth the following criteria for interpretation of WB results:
Supported Bands:
- Bands corresponding to the predicted size in kDa (+/-20%).
- Band of predicted size in kDa (+/-20%) with additional bands present.
Uncertain bands:
- Single band larger than predicted size in kDa (+20%) but partly supported by predicted transmembrane region, signal peptide or by other available data.
- No bands detected.
- Single band differing more than +/-20% from predicted size in kDa and not supported by predicted transmembrane region, signal peptide or by other available data.
- Weak band of predicted size in kDa (+/-20%) but with additional bands of higher intensity also present.
- Only bands not corresponding to the predicted size.
- Target too small/large to be analyzed with the present setup.
- Current setup is not applicable due to low RNA count
https://www.proteinatlas.org/about/antibody+validation
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