Hello everybody!
I'm trying to analyze spermatozoa protein expression but I have some problems.
I can lyse spermatozoa only with leammly (10', 95 C°): so, it's impossible for me to quantify previously protein concentration.
I need to compare cases versus control.
This is my idea:
- to lyse more or less the same number of cells
- for each sample, normalize the expression of my protein of interest with GAPDH expression
- then compare my cases versus the average signal of my controls
Do you think it could be rational?
Thank you :)
Based on my little experience, I would say it is ok.
Normalization on housekeeping protein (e.g. GAPDH) is used exactly as an internal loading control; loading equals amount of total proteins extract in each gel lanes could be a further control on the housekeeping protein reliability.
In your case I would say you must be sure that GAPDH is a good housekeeping protein in your experimental conditions cause you will not have this further control.
And what about using a pre-antibody reversible staining (such as Ponceau S) to control amount of loaded proteins and use it for normalization? Here a reference.
SDS-PAGE and western blot are used mainly for qualitative detection of proteins for their expression levels. Do not you use other protein assay procedures for quantification?
Unfortunately we can't because we lysis human sperm directly in Laemmly
For relative quantification the main point is to find a proper reference. Sperm count is a good start. Next consideration is an internal reference, such as a housekeeping protein (or even better, more than one). You should try to chose one or more proteins that are not affected by the conditions you are studying.
About reference and housekeeping proteins: you ALWAYS need to validate them, as Giorgio Medici pointed out. Do not just trust what other people are using. GAPDH sure is a candidate, but I suggest you also test other ones, such as tubulin, actine, mitocondrial proteins and protamines. I suggest you do:
- sperm count to know you have the same sperm concentration in your samples before lysis
- load same amount per samples (control vs cases)
- test the prospective reference proteins you have chosen
- quantify each protein band and finally pick the one(s) that show less variation between the samples
At the same time you can also try Ponceau to see if it works for you.
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