Hi every one,
My PI and i are performing Western blot on B Amyloid protein. Unfortunately, after developing the Membrane, the control protein we are using (Tubulin) does not appear to have standard bands throughout all the wells. What could be the problem, has any body faced a similar dilemma where everything else appears okay but the Tubulin bands have different intensities in different samples wells?
Hi,
I am sorry to tell you this but control proteins or house keeping proteins (HKP) are not valid. This has mainly two reasons: 1. Every protein in a cell is regulated. Over 30 paper will tell you that commonly used HKP are regulated depending on treatment or cell type or cell age or...... In most Western blots this regulation is just not detectable but present in the cell. This makes these proteins to an invalid normalization factor. 2. For your Western blot you normally load enough protein to detect your actual target. This means that you have loaded a high amount of your HKP as these are high abundant. This leads to a saturated membrane and a HKP-signal that is way beyond linearity. You can see differences in your HKP-signal but you have no idea if you have a regulated HKP or an artifact in your blot. Either way you can not use this signal as a loading control.
Protein staining is a better way to normalize your target signal but if you have excess to a fluorescence imager a fluorescence labeling of your total protein will give you the best normalization factor. This is because you are detecting the total protein and your target at the same time. This way you can also eliminate signal variations caused by protein loss during the washing, blocking and antibody binding procedure.
For more information please have a look on the following website:
https://www.dyeagnostics.com/site/en/products/spl/
Best regards,
Kristin
This could be an artifact of unequal loading, blocking or washing steps. You can repeat your experiment taking more precaution in those steps and see if the issue will be resolved. The advice given by Kristin above on the best normalisation option is another good way to resolve this issue.
Best wishes,
Aminu.
Some scientists load lysates based on equal number of cells, others load equal protein content per lane. It depends on the objectives of the experiment. Either way, a quick ponceau stain after transfer is required to assess the quality of transfer (mark air bubbles, dismiss lanes where transfer failed at positions of interest) and to confirm that all lanes contain similar or equal protein content. Record the image before washing off the ponceau, then continue with the immunolabeling. You still need a loading control antibody though. When tubulin remains variable, try a different HKP for example GAPDH or beta Actin.
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