yes u just centrifuge the crude extract and remove the pallet.....after that u can calculate the total protein by different protein estimation methods...however the proteins are in mixture and u couldnt get the actual concentration of particular protein for it u have to go for purification and if u know the molecular weight of ur protein then u can see it on SDS page by loading this crude sample on gel this is just for checking the presence of your protein in sample

 if i'll discard the pellet i might loose the protein, if my protein is present in the pellet fraction.

so don't know whether i use this method or not.

if i can use this method in this position also?

can i measure the protein concentration by loading a standard protein (known conc.) adjacent to my protein on SDS-PAGE?

As u told that u want to know the protein in cel extract.....then this method can be used...tell me one thing as ur protein is extracellular or intracellular...if u know it then u can directly do this experiment

and if u didnt know the exact nature of protein then do one thing dont discard the pallet and have reaction with substrate by adding pallet and extract  into two different tubes. it will show the presence of ur sample...

ok, means i can use pellet also with bradford method?

or i can only use bradford with supernatent part?

You can use LOWRY’S METHOD for this which is very simple and not costlier for protein determination. See attachment for detail procedures.

Well, you can use Bradford method or Lowery Method for both of your fractions.

In another simple method you can precipitate all of your proteins (soluble or insoluble) by ammonium sulfate and can quantify the protein contents by any method.

You can also use another method, whereby calculate the weight of your liquid. Then freeze dry your sample (measure its weight) and analyze the protein contents by Dumatherm or Kjeldhal Method. Then by dividing the concentration factor from liquid to solid you can have your protein contents in your sample.

Hope this will help

Both Lowry and Bradford methods can be used.

Lowry method is approximately harder and need several steps but Bradford is easy and can perform in few steps. Also, the excision of Bradford method is 1000 times more than Lowry method (ng vs. ug).

Thanks for the suggestions. 

You can also apply BCA assay which tolerates detergents and is as easy as Bradford's (expensive but worthy, half bottle last for 3 yrs and not used up...) if yours a membrane protein.

Bear in mind that this method has no end point so you need to complete the determination within short time or else lead to erroneous results.

First of all, to figure out whether the protein exist in pellet fraction or in supernatan fraction, you might want to try to read the supernatan fraction Absorbance in 280nm directly using quartz cuvette. I think it's the most simple way and it provide you the basic knowledge you need to know about your protein. Because if it's extracellular, you'll find it in your supernatan. And since you measure the supernatan directly, you can always recovered your sample for further protein assay method such as Lowry, Bradford, or SDS PAGE

Hope this help. 

Yes i agree with Christine Dwi A P W....but for this u must know the nature of ur protein that is weather it is in supernatant or in pallet......so perform enzyme reaction (Enzyme +substrate) with both fraction and conclude ur result that were the  protein is actually present the follow the procedure of absorption at 280 nm

As for me, i'd first carry out SDS-PAGE to find out its nature of target protein, also you can acquire it from articles, rather than A280 or any other colorimetric method if your goal is to get the pure protein. It's a simple and cost-effective way to determine.

Like this: M, (soluble in native), (pellet in denaturing w/ deter), (pellet in dena w/o deter)...as one's wish

The reason why I don't use A280 beforehand is because this only detects residues with aromatic side chains (W>>Y>F, H no, see links below) and Cys, a good example is human ubiquitin having no Ws, Cs but 1Y &1F therefore hard to detect (almost background).

Next, go on a strategic planning and get parameters like purification fold, yield, etc. from lysate w/ or w/o deter & denaturing buffer. If yours is soluble, things get simple and vice versa.

http://www.wikiwand.com/en/Amino_acid#/Table_of_standard_amino_acid_abbreviations_and_properties

http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aromatic.html

I do agree with Penn's opinion on carrying out SDS-PAGE, but my view is this should be done on both the supernatant and the pellet. Followed by crude protein determination using Bradford reagent.