I'm working with a neuron cell line in culture and I will want to check the levels of different extracellular proteins using western blot
Hi Tatiana,
I think that to quantify secreted proteins to the cell medium, the best way is the use of ELISA. However, you can use WB following the protocol the authors used in this paper: DOI 10.1099/mic.0.2006/000513-0
Good luck!
Hi Tatiana,
I think that to quantify secreted proteins to the cell medium, the best way is the use of ELISA. However, you can use WB following the protocol the authors used in this paper: DOI 10.1099/mic.0.2006/000513-0
Good luck!
Hi Tatiana - probably the biggest problem you are facing is the volume of the media that you have (to much to load onto a Western blot). If you want to avoid using an ELISA you could concentrate up the samples using a protein concentrator (such as this:
http://www.piercenet.com/product/pierce-protein-concentrators-20k-mwco)
If you have a rough idea of the size of the protein you can choose the column that you need (or indeed run several different columns). There are quite a few types around (mostly centrifuge based) the ones from Pierce are just an example (I have no affiliation to Pierce). I used to use Viva spin columns:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/28932218
Hope this helps,
Gary
Hello Tatiana,
i do agree with both excellent suggestions above.
it always depends on the abundance of the protein you are after and also your budget
some alternative suggestions
1. One option is to start with ELISA, sometimes you can get an ELISA array plate custom made just to identify what proteins are present in your material of interest and then you can expand the analysis with western blotting if you prefer for specific targets.
You could also make your own coated plate provided you have the proteins of interest and the relevant antibodies for detection.
2. Alternatively if you know your targets you could try protein immunoprecipitation (IP) which will allow you to pull down specific proteins and be able to concentrate your samples as much as you want.
you can pull down multiple proteins by re-using the same extract so in this way you dont waste material.
3. a more expensive analysis is to try mass spec
hope that helps
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