Dear researchers,
I will look at ER stress proteins from rat liver tissue by Western blot method. I found the my protein concentration to be 86.386 µg / ul. How many ug of protein should I choose accordingly? How many ul of Laemmli Buffer Solution (2x) should I add for loading? Is the concentration of the liver lower compared to other tissues? Should I be careful about this when determining the protein amount?
Thank you.
Hi Didem,
First of all, protein content of liver is pretty high compared to many other tissues. So you should be careful when preparing the tissue lysate (to be sure you properly lysed all membranes).
It looks like your protein concentration ~86 ug/ul is really high. I don't know which method you used to detect the concentration but it is highly above the working range of many methods, so I hope you diluted your sample before measuring the concentration. I would suggest you simply take some from your sample and dilute it 5 times and 10 times and measure it. Then multiply what you found with the dilution factor (5 or 10). If the concentration is correct, I suggest you dilute your sample in your lysis buffer, because you don't want to load 1 ul to the gel, that increases pipetting errors.
You can load as low as 1 ug or as high as 50 ug protein. That depends on your target protein. For example; for the proteins like Histone-H3, GAPDH, Tubulin etc (excess proteins in cells and tissues) you can load 5 ug that should be more than enough. But if you need to detect phosphorylated protein or a protein which as very low abundance, you need to be loading more like 30 ug.
Let's say your concentration is 10 ug/ul, and you decided to load 50 ug to the gel. You will need 5 ul from your sample and you will mix it with the same amount of Laemmli solution. 2X concentration of Laemmli will be 1X at the end.
Hello Sibel Cal-Kayitmazbatir I wanted to ask you regarding western blot please, I am actually using western blot currently to detect the expression of XPC protein in keratinocyte cell lines and I am using a fibroblast cell line that does not express XPC as a negative ctrl, upon the normalization with GAPDH I found non specific band, I loaded 42,4 micrograms of proteins. Furthermore, I was also looking for p53 expression and I also used 42.4 micrograms and the results appeared like that of GAPDH, the arrows in the p53 image corresponds to a well with water and lamelli and the second one was empty...please do you have any advice?
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