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Protein Quantification

Assays

What It Is  Protein quantification is the measurement of the amount of protein in a sample  Currently a vast number of ways of quantifying proteins  3 Main methods  Spectroscopic Procedures  Measurement through colorimetry  Amino acid analysis  Other methods (radiolableling. RP-HPLC) . degradation.

and stable for up to 1 hour  Challenges: requires proteins larger than 3 kDa. SDS & Triton X-100) . simple. is sensitive to detergents (eg.Bradford Assay  Colorimetry assay  Negatively-charged dye solution (Red in solution – absorbs @ 465 nm)  Binds to positively-charged proteins (Turns blue – absorbs @ 595 nm)  Reaction is fast.

Folin-Lowry Assay  Colorimetry Assay  Complexes Cu with N in the protein  Complexed Tyr and Trp react with Folin Ciocalteu phenol reagent  Yeilds a solution with blue-green color (absorbs @ 650-750 nm)  Stable result. can estimate protein from one assay by comparing to a standard curve  Incompatible with many common chemicals .

nucleic acids) .Ultraviolet Absorption Assay  Measures characteristic absorption of Tyr and Trp at 280 nm  Challenges  Every protein has a different amount of these amino acids  Many molecules interfere at 280 nm (alcohols. buffers.

Kjeldahl  Extremely old method (133 years)  Measures N in a protein sample post conversion to ammonia   Heated sulfuric acid. back-calculate total amount of protein Challenges: time-consuming. back-titration with NaOH  Weigh out the purified N  Assume 16% N in original sample. requires at least 1g of sample . steam distillation.

med.gov/pubmed/942051  http:// research.ncbi.nih.helsinki.nlm.fi/corefacilities/proteinchem/Protein%20quanti fication%20and%20detection%202012.com/23824/top-5-protein-quantification-assays/  http://www.pdf .References  http://bitesizebio.