Bradford Assay
What is the broad range of detection for the Bradford assay spectrophotometer? What is the minimum protein size?
-1 to 2000 ug/mL -3000 to 5000 dalton so (3-5 kDa)
What are the cons of Bradford assay?
-binding is hindered by several reagents that may be present in the assay like detergents, biological buffers, sugars, etc. -cannot determine single type of protein [] in mixed sample
What is a Bradford Assay? And what is a key component of material that you use?
-colorimetric assay -Coomassie brilliant blue G250 dye -helps to find the total protein concentration
How does Coomassie brilliant blue G250 interact with the protein
-dye binds to exposed surfaces of basic (histidine, lysine, arginine) and aromatic AAs (phenylalanine, tyrosine, and tryptophan)
What is Coomassie brilliant blue G250, and what does it do?
-dye that is kept at a low pH in green form; mixture the Bradford solution alone is brown -formation of the protein/dye complex stabilizes the negatively charged anionic form of the dye producing the blue color
What are the pros of Bradford assay?
-fast -one step procedure -inexpensive -sensitive -highly specific for protein
What are some applications that can be used from using the Bradford assay?
-food protein analysis -protein purification -comparing samples
What is a colorimetric assay and what does it do?
-measures color change relative to standard (color) -allows for quantification through spectrophotometry
What is the purpose of quantifying a protein using the Bradford Assay?
-proteins are central to our understanding of biology -in immunological cells- they are multipurpose ---> TCR and MHC activating T cells ---> Modulating signal transduction pathways: kinases ---> antibodies identifying invaders
What is the purpose of the Bradford Assay?
-quantitative protein measurements -standard curve -determination of an unknown protein concentration
What is the significance of the color change of the dye?
Color change is directly proportional to the amount of protein -lighter = less protein amount -darker = more protein amount
When reading absorbable, why should you avoid bubbles in your samples?
The light beam will pass through air instead of your sample solution
