I loaded 10ul of raw seminal plasma sample + 10 ul Bolt LDS buffer +4ul Bolt reducing reagent. I did the transfer using iBlot2 dry transfer system and used One Pierce ECL protein detection kit. The resulting blot was viewed using iBright fL1000. The problem is that i didnt use a housekeeping gene.
If you are relatively confident that your sample prep for the seminal plasma is reproducible, then you have actually 'normalized' by method -- and perhaps you don't really need to worry about normalizing on the blot itself.
As a possible alternative, you might normalize to total protein concentration in the crude samples (assuming you have any volume remaining). Many studies assume that total protein from any particular cell/tissue should be a relatively constant metric which can be used to normalize. There are caveats of why this simple approach might not be accurate for any specific experiment, but it's a starting point. No matter how you normalize, it's wise to spend a little extra effort proving to yourself that indeed your normalization metric indeed is invariant across your samples.
if you have access to BioRad's ChemiDoc system you can actual use stain free gels, UV cross link them, and then measure total protein transferred. You could normalize your data to that. I would not assume that just because you load the same sample volume that you are loading equal protein levels. Can you ran a total protein assay such as Pierce's BCA assay?
In order to ensure you are loading equal concentration of each sample, you must perform a protein quantification assay. Equal volume wont give equal concentration. Consider to do BCA or bradford assay.
Good luck.
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