COMPARISION OF DIFFERENT PROTEIN DETERMINATION METHODS

作者：Protocol    实验频道来源：本站原创    点击数：    更新时间：2004-7-21

COMPARISION OF DIFFERENT PROTEIN DETERMINATION METHODS Company Method Detection  Range Applications -Compatibility Assay protocol Precautions-Interferences Absorbance 280nm 0.1-1 OD280nm/ml Absorbance of aromatic amino-acids (Trp, Tyr and Phe at less extent). Only for purified proteins with known absorptivity factor (Use ExPASy ProtParam tool to inquire E1% 280nm) Read absorbance 280nm. Nucleic acid, detergents, cofactors, phenolic compounds, pigments, reducing agents, etc etc. AMERSHAM- BIOSCIENCE (PHARMACIA) (# 80-6483-56) 2-D Quant Kit  0–50 µg (1–50 µl)  Designed for the accurate determination of protein concentration in samples prepared for electrophoresis and presence of detergents, Urea, DTT, EDTA, Ampholites, etc, and many buffer components. Quantitative precipitation of proteins while leaving interfering substances behind.  BIO-RAD (#500-0001/2/6) Bio-Rad Protein Assay  (Modified  Bradford) (pdf) 0.2–0.9 mg/ml Compatible with reducing agents  (See list of compatible  reagents on BioRad cataloge) Minimum incubation time 15minutes. Assay wavelength 650-750nm Detergents, basic buffers BIO-RAD (#500-0111/2) DC Protein Assay (Modified Lowry) (pdf) 0.1–2.0 mg/ml Compatible with detergents, basic buffers (See list of compatible reagents on BioRad cataloge) Minimum incubation time 15minutes. Assay wavelength 595nm Reducing agents BIO-RAD (#500-0121/2) RC DC Protein Assay (Modified Lowry) (pdf) 0.1–2.0 mg/ml Compatible with detergents, reducing agents, Laemmli sample buffer with 5% beta-mercaptoethanol, etc (See list of compatible reagents on BioRad cataloge) Minimum incubation time 15minutes. Assay wavelength 650-750nm GENO TECHNOLOGY (#786-005) Non-Interfering Protein AssayTM Compatible with reducing agents(2ME, DTT), chelating agents EDTA , detergents (non-ionic, anionic, cationic, and zwitterionic) , amines (Tris), sugar, urea, ammonium sulfate, guanidine hydrochloride, guanidine thiocyanate, drugs, antibiotics, cobalt, and numerous other agents. Quantitative precipitation of proteins while leaving interfering substances behind. MOLECULAR PROBES  (N-6666) Nano Orange Protein Quantitation Kit (pdf) 10 ng/mL -  10 µg/mL Little protein-to-protein variability. Compatible with the presence of reducing agents and nucleic acids.  Mix and heat 10' 95ºC. Fluorescence emissions are measured directly Unusually high concentrations of lipids in the sample can interfere. This interference can be eliminated by acetone precipitation of the protein, followed by delipidation with diethyl ether. MOLECULAR PROBES  (C-6667) CBQCA Protein Quantitation Kit (pdf) 10 ng/mL - 150 µg/mL Functions well in the presence of lipids and detergents (to determine the protein content of lipoprotein samples or lipid–protein mixtures) PIERCE (#23225) BCA Protein Assay (pdf) 0.5-20 µg/ml Compatible with detergents solubilized proteins. Proteins on affinity supports. Chaotropic agents, sugars, DNA, protease inhibitors 2ml reagent + 0.1ml sample Incubation 30' 37ºC. Read 562nm Reducing agents (can be eliminated with TCA, see Protocol) PIERCE (#23235) Micro-BCA Protein Assay (pdf) 0.5-20 µg/ml Compatible with detergents solubilized proteins. Proteins on affinity supports. Chaotropic agents, sugars, DNA, protease inhibitors 1ml reagent + 1ml sample Incubation 60' 60ºC. Read 562nm Reducing agents (can be eliminated with TCA, see Protocol) PIERCE (#23236) Coomassie Plus Protein Assay 1-1500 µg/m For quick estimation where accuracy is not important. Reducing agents. Chaotropic and chelating agents, metals, protease inhibitors. DNA. 3 ml reagent + 0.1ml sample Vortex and read 595nm Detergents (sometimes you can normalize using same detergent concentration in blank and standarts) PIERCE (#23200) SIGMA (#610-A) Coomassie Protein Assay (pdf) BRADFORD 1-1500 µg/ml For quick estimation where accuracy is not important: great variability. Compatible with reducing agents. Chaotropic and chelating agents, metals, protease inhibitors. DNA. 5 ml reagent + 0.1ml sample Vortex and read 595nm Detergents (sometimes you can normalize using same detergent concentration in blank and standarts) PIERCE (#23240) Modified Lowry Protein Assay (pdf) 1-1500 µg/ml Accurate. Compatible with protease inhibitors. DNA. 1 ml reagent + 0.2ml sample. Incubate 10' RT. Add 0.1ml Folin-Ciocalteu. Incubate 30' RT. Read 750nm Reducing agents (can be eliminated with TCA, see Protocol,pdf) and some detergents PIERCE (#23255) Fluoraldehyde Protein/Peptide Assay 0.05-500 µg/ml Compatible with reducing agents and some detergents. Chelating agents, metals and sugars 2 ml reagent + 0.2ml sample. Mix and read fluorescence; excitation 330-390nm; emission 436-475nm. Great variability. Cannot meassure in the presence of TRIS or Glycine buffer. ROCHE  (#1 767 283) (#1 767 003) ESL Protein Assay  (pdf) 20-800 µg/ml. Detection limit 1µg/sample Biuret-like reaction, detect peptide bonds. The assay is compatible with several detergents. Coul be used for determination of peptides and immobilized proteins 7 minutes reaction. Read at 485nm. SIGMA (#690-1) BIURET 1-10 mg/ml Very accurate and simple Read 550nm Glucose, Ammonium sulphate, Sulphydryl compounds, PO4 buffers Folin-Ciocalteu reagent: SIGMA (#F9252) - MERCK LOWRY 10 µg/ml - 1 mg/ml Very accurate 1 ml reagent + 0.2ml sample. Incubate 10' RT. Add 0.1ml Folin-Ciocalteu. Incubate 30' RT. Read 750nm Many detergents, reducing agents, EDTA, GuHCl, AmmSO4, >0.1M TRIS. Interfernce elimination with TCA or DOC-TCA SIGMA (#P5656) Folin-Ciocalteu reagent: SIGMA (#F9252) - MERCK LOWRY-PETERSON 1-10 µg protein Modified Lowry for membrane proteins. Compatible with detergents and with the presence of all kind of interferents that can be eliminated by DOC-TCA precipitation. DOC-TCA precipitation of proteins before Lowry assay BUTTERFLY (coomassie staining into 3MM Whatman paper) Very useful technique for proteins in all kind of solutions (like PAGE-SDS sample buffer) Dry samples into 3MM Whatman paper.Treate with coomassie staining solution for ~30 min. Distain. Extracted ON with 3% SDS solution and read at 590 nm 处理 SSI 文件时出错