COMPARISION OF DIFFERENT PROTEIN DETERMINATION METHODS

互联网2013-09-05

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<center> COMPARISION OF DIFFERENT PROTEIN DETERMINATION METHODS </center>

<center> <table> <tbody> <tr> <td> Company </td> <td> Method </td> <td> Detection Range </td> <td> <center> Applications -Compatibility </center> </td> <td> Assay protocol </td> <td> <center> Precautions- </center> Interferences </td> </tr> <tr> <td> </td> <td> Absorbance 280nm </td> <td> 0.1-1 OD280nm/ml </td> <td> 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) </td> <td> Read absorbance 280nm. </td> <td> Nucleic acid, detergents, cofactors, phenolic compounds, pigments, reducing agents, etc etc. </td> </tr> <tr> <td> AMERSHAM- BIOSCIENCE (PHARMACIA) (# 80-6483-56) </td> <td> 2-D Quant Kit </td> <td> 0�50 µg ( 1�50 µl) </td> <td> 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. </td> <td> Quantitative precipitation of proteins while leaving interfering substances behind. </td> <td> </td> </tr> <tr> <td> BIO-RAD (#500-0001/2/6) </td> <td> Bio-Rad Protein Assay (Modified Bradford) (pdf) </td> <td> 0.2�0.9 mg/ml </td> <td> Compatible with reducing agents (See list of compatible reagents on BioRad cataloge) </td> <td> Minimum incubation time 15minutes. Assay wavelength 650-750nm </td> <td> Detergents, basic buffers </td> </tr> <tr> <td> BIO-RAD (#500-0111/2) </td> <td> DC Protein Assay (Modified Lowry) (pdf) </td> <td> 0.1�2.0 mg/ml </td> <td> Compatible with detergents, basic buffers (See list of compatible reagents on BioRad cataloge) </td> <td> Minimum incubation time 15minutes. Assay wavelength 595nm </td> <td> Reducing agents </td> </tr> <tr> <td> BIO-RAD (#500-0121/2) </td> <td> RC DC Protein Assay (Modified Lowry) (pdf) </td> <td> 0.1�2.0 mg/ml </td> <td> Compatible with detergents, reducing agents, Laemmli sample buffer with 5% beta-mercaptoethanol, etc (See list of compatible reagents on BioRad cataloge) </td> <td> Minimum incubation time 15minutes. Assay wavelength 650-750nm </td> <td> </td> </tr> <tr> <td> GENO TECHNOLOGY (#786-005) </td> <td> Non-Interfering Protein AssayTM </td> <td> </td> <td> 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. </td> <td> Quantitative precipitation of proteins while leaving interfering substances behind. </td> <td> </td> </tr> <tr> <td> MOLECULAR PROBES (N-6666) </td> <td> Nano Orange Protein Quantitation Kit (pdf) </td> <td> 10 ng/mL - 10 µg/mL </td> <td> Little protein-to-protein variability. Compatible with the presence of reducing agents and nucleic acids. </td> <td> Mix and heat 10' 95ºC. Fluorescence emissions are measured directly </td> <td> 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. </td> </tr> <tr> <td> MOLECULAR PROBES (C-6667) </td> <td> CBQCA Protein Quantitation Kit (pdf) </td> <td> 10 ng/mL - 150 µg/mL </td> <td> Functions well in the presence of lipids and detergents (to determine the protein content of lipoprotein samples or lipid�protein mixtures) </td> <td> </td> <td> </td> </tr> <tr> <td> PIERCE (#23225) </td> <td> BCA Protein Assay (pdf) </td> <td> 0.5-20 µg/ml </td> <td> Compatible with detergents solubilized proteins. Proteins on affinity supports. Chaotropic agents, sugars, DNA, protease inhibitors </td> <td> 2ml reagent + 0.1ml sample Incubation 30' 37ºC. Read 562nm </td> <td> Reducing agents (can be eliminated with TCA, see Protocol) </td> </tr> <tr> <td> PIERCE (#23235) </td> <td> Micro-BCA Protein Assay (pdf) </td> <td> 0.5-20 µg/ml </td> <td> Compatible with detergents solubilized proteins. Proteins on affinity supports. Chaotropic agents, sugars, DNA, protease inhibitors </td> <td> 1ml reagent + 1ml sample Incubation 60' 60ºC. Read 562nm </td> <td> Reducing agents (can be eliminated with TCA, see Protocol) </td> </tr> <tr> <td> PIERCE (#23236) </td> <td> Coomassie Plus Protein Assay </td> <td> 1-1500 µg/m </td> <td> For quick estimation where accuracy is not important. Reducing agents. Chaotropic and chelating agents, metals, protease inhibitors. DNA. </td> <td> 3 ml reagent + 0.1ml sample Vortex and read 595nm </td> <td> Detergents (sometimes you can normalize using same detergent concentration in blank and standarts ) </td> </tr> <tr> <td> PIERCE (#23200) SIGMA (#610-A) </td> <td> Coomassie Protein Assay (pdf) BRADFORD </td> <td> 1-1500 µg/ml </td> <td> For quick estimation where accuracy is not important: great variability . Compatible with reducing agents. Chaotropic and chelating agents, metals, protease inhibitors. DNA. </td> <td> 5 ml reagent + 0.1ml sample Vortex and read 595nm </td> <td> Detergents (sometimes you can normalize using same detergent concentration in blank and standarts ) </td> </tr> <tr> <td> PIERCE (#23240) </td> <td> Modified Lowry Protein Assay (pdf) </td> <td> 1-1500 µg/ml </td> <td> Accurate. Compatible with protease inhibitors. DNA. </td> <td> 1 ml reagent + 0.2ml sample. Incubate 10' RT. Add 0.1ml Folin-Ciocalteu. Incubate 30' RT. Read 750nm </td> <td> Reducing agents (can be eliminated with TCA, see Protocol , pdf ) and some detergents </td> </tr> <tr> <td> PIERCE (#23255) </td> <td> Fluoraldehyde Protein/Peptide Assay </td> <td> 0.05-500 µg/ml </td> <td> Compatible with reducing agents and some detergents. Chelating agents, metals and sugars </td> <td> 2 ml reagent + 0.2ml sample. Mix and read fluorescence; excitation 330-390nm; emission 436-475nm. </td> <td> Great variability. Cannot meassure in the presence of TRIS or Glycine buffer. </td> </tr> <tr> <td> ROCHE (#1 767 283) (#1 767 003) </td> <td> ESL Protein Assay (pdf) </td> <td> 20-800 µg/ml. Detection limit 1µg/sample </td> <td> Biuret-like reaction, detect peptide bonds. The assay is compatible with several detergents. Coul be used for determination of peptides and immobilized proteins </td> <td> 7 minutes reaction. Read at 485nm. </td> <td> </td> </tr> <tr> <td> SIGMA (#690-1) </td> <td> BIURET </td> <td> 1-10 mg/ml </td> <td> Very accurate and simple </td> <td> Read 550nm </td> <td> Glucose, Ammonium sulphate, Sulphydryl compounds, PO4 buffers </td> </tr> <tr> <td> Folin-Ciocalteu reagent: SIGMA (#F9252) - MERCK </td> <td> LOWRY </td> <td> 10 µg/ml - 1 mg/ml </td> <td> Very accurate </td> <td> 1 ml reagent + 0.2ml sample. Incubate 10' RT. Add 0.1ml Folin-Ciocalteu. Incubate 30' RT. Read 750nm </td> <td> Many detergents, reducing agents, EDTA, GuHCl, AmmSO4, >0.1M TRIS. Interfernce elimination with TCA or DOC-TCA </td> </tr> <tr> <td> SIGMA (#P5656) Folin-Ciocalteu reagent: SIGMA (#F9252) - MERCK </td> <td> LOWRY-PETERSON </td> <td> 1-10 µg protein </td> <td> 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. </td> <td> DOC-TCA precipitation of proteins before Lowry assay </td> <td> </td> </tr> <tr> <td> </td> <td> BUTTERFLY (coomassie staining into 3MM Whatman paper) </td> <td> </td> <td> Very useful technique for proteins in all kind of solutions (like PAGE-SDS sample buffer) </td> <td> 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 </td> <td> </td> </tr> </tbody> </table> </center>