I am using the following protocol for measuring rubisco content:
Large subunits and relevant small subunits were cut and washed
with 3 mL of formamide in a 10 mL cuvette, then incubated
in a 50 °C constant temperature water bath for 8 h. The
washed solutions were analyzed via spectrophotometry at
a wavelength of 595 nm, using the background gel as blank
and bovine serum album (BSA) as protein standard.
Does it mean Bradford assay? or do I check the absorbance of BSA standards prepared in distilled water only?
protein determanation spectrophotometry at a wavelength of 595 nm is used in Bradford assay but in this method BSA is not a good standard and you most change it .
In this instance, I think that the protein concentration is being measured in the following way. The RuBisCO protein is run on a gel, along with various amounts of BSA for standards. The gel is stained with Coomassie Blue. The stained bands are cut out of the gel. The stain is eluted from the gel using formamide as a solvent. The amount of eluted stain is measured by its absorbance at 595 nm using a spectrophotometer.
One other question, I have performed Bradford assay for protein quantification using 0.5 g of fresh tissue and 2 ml of the extraction buffer. I have my values in mg/ml, how would I express them in mg/g?
(X mg protein/ml of extract) x (2 ml of extract/0.5 g of tissue) =
4X mg of protein/g of tissue.
In other words, multiply mg/ml by 4.
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