Could anyone suggest a protocol of extracting nuclear protein and detecting the protein's concentration? I mean, I'd like to get respective nuclear and cytoplasmic protein from our -80 degree stored mouse kidney tissues. Plus, is there any different from the extraction of cytoplasmic protein only? Thanks for your help.
Life technology sells a kit for this and also find the protocol by clicking on the below link. If you can make the reagents in-house- that'd save you some money.
https://www.lifetechnologies.com/uk/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/nuclear-extraction-method-.html
Re conc. of the protein- it all depends on how much protein you're expecting and accordingly you may measure using various methods. However I'd guess the conc. of protein would probably be very low- therefore first I'd like to test it using an antibody like western blot.
Calbiochem (through Millipore) also sells a fairly reliable kit (cat #539790-1KIT) for extracting proteins based on their subcellular location. I would suggest using their "Non-Interfering Protein Assay" kit (cat #488250) for determining protein concentration. They also sell a protein precipitation kit (cat#539180) that might be useful if the protein concentration is too low to detect after extraction.
In principle it should. you may have to homogenise the tissue and then go for cell lysis. there are also kits which you can use for this purpose.
That tells you that the amount of protein you've got in your sample is not enough to be detected by this assay. you may have to try a more sensitive method for this.
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