Wednesday, January 30, 2013
Protein Quantification (Bradford Assay)
Bradford assay is a rapid and accurate method to determine the concentration of protein. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Within the linear range of the assay (~5-25 mcg/ml), the more protein present, the more Coomassie binds.
Bradford Assay
Experiment using Bradford Assay to determine protein concentration. Follows a specific protocol but can be used to understand how to perform a Bradford Assay.
How to Make an SDS-PAGE gel
Alright so here's a quick video on how to cast an SDS-PAGE gel. Although recipes can vary, the ingredients shown here are almost always used. Remember: always add TEMED last, and pour into plates immediately after!
Already know how to make a gel, but have problems with leaky gels? Check out our other video on How to Avoid a Leaky SDS-PAGE gel: See the video here
Quantitative estimation of aminoacids by Ninhydrin Method
Amino acids are known to be the building block of all proteins. Twenty different amino acids are commonly found in proteins. Amino acids comprises of a carboxyl group and an amino group bonded to the same carbon atom (the α carbon).They differ from each other in their side chains (R groups),which vary in structure, size, electric charge and solubility in water. Hence their detection, quantification and identification in any sample constitute important steps in the study of proteins.
