I am very new to western blotting and I am not really sure about how to set up my western blot. I have a treatment and a control group, each with around 7 samples from the eyes of chicks. I want to observe the difference in expression of a protein between the two groups.
I am going to use beta-tubulin for a HKP, however I have been reading that total protein staining may be a better option for normalization.
My main problem is that I don't know what I should include on each gel I run. I am using 10-well precast gels.
Would it be a good idea to pool the samples and run them, or should I run the samples individually and analyze them that way?
Also, what would be the best method for comparing results between gels?
Any help would be greatly appreciated,
Thank you
Hi Nicholas,
if you want to quantify something, perform an ELISA with your individual samples,
that would be the method of choice for your Experiment.
Hi Nicholas,
I think that a good method for comparing results between gels is to load a control sample in both of them (same amount of protein on both), and then normalize the signal of each lane to that of the control (in each gel). Also, I think it's better to run and analyze your samples individually.
Always load protein (15-20ug, must equal in each well) from control and treatment alternatively in the single gel (Control1, treatment 1…..etc) and then detect the protein expression of housekeeping and target proteins. Further, normalize with housekeeping protein by image J software. That would help you to get clear protein expression
Better idea to make the higher concentration of protein per millilitre when you are extracting protein from samples (2-3mg/ml or >). Then you can use 15 well SDS-PAGE.
Hi Nicholas,
welcome to the world of Western blotting. To get reliable results normalization is in deed the most important step. HKP are not the best option as these can be regulated without beeing noticed as they are mostely very high abundend and saturate signal and membran. Regulations that are present in your cell are not translated on your blot und you will get unreliable results. To use the total protein gives you thousends of proteins that gives you a much better statistic. Visualization of the total protein should be at the time of target detection though. This can only work using fluorescent protein labeling. But the total protein alone does not give you all answers about possible variations in the Western blotting process. Therefore you need a sample specific standard.
This sounds like a lot of work, I know. But If you want the total control and the most reliable results I can only recommend this method. A ready to use kit with all labels and standards is available at:
https://www.dyeagnostics.com/site/en/products/spl/
I would be happy to help you with your first Western blots. With this system even normlization between different blots is possible, if you have to split your samples using 10 well gels. Contact me if you have questions.
Best regards,
Kristin
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