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Bradford/ BioRad Protein Assay

Bradford/ BioRad Protein Assay. Biol 3018 – Investigaci ó n JA Carde. Materiales. Biorad Protein Assay : ( Biorad Protein Dye , cat # 500-0006) – JA Cardé Previamente preparar: Espectrofotómetro encendido en  595nm Bovine Serum Albumin (BSA) 0.5-1ug/ ul

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Bradford/ BioRad Protein Assay

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  1. Bradford/BioRad Protein Assay Biol 3018 – Investigación JA Carde

  2. Materiales • BioradProteinAssay: (BioradProteinDye, cat # 500-0006) – JA Cardé • Previamente preparar: • Espectrofotómetro encendido en  595nm • BovineSerumAlbumin (BSA) 0.5-1ug/ul • BioradProteinDye (cat # 500-0006), diluido 1:5 con agua dd, vortex. (dura 2 semanas a RT) • Cuvetas de espectrofotometría (1ml, 1cm path, polipropileno, semimicro, Biorad # cat. 223-9950) • Micropipetas de 1000ul, 100ul, 10ul, 1ul • Kim wipes • Susmuestras en hielo

  3. Introduccion • Four spectroscopic methods are routinely used to determine the concentration of protein in a solution • Measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; • Lowry assay • Smith copper/bicinchoninicassay • Bradford dye assay. • Although one or more these methods is used routinely in almost every biochemical laboratory, none of the procedures are particularly convenient, for the reasons described below.

  4. Bradford Assay • Based on the equilibrium between three forms of Coomassie Blue G dye.  • Under strongly acid conditions, the dye is most stable as a doubly-protonated red form. • Upon binding to protein, however, it is most stable as an unprotonated, blue form. • Red Green  Blue-Protein (470 nm)          (650nm)   (590 nm)  H+      H+

  5. Bradford • The Bradford assay is faster • Involves fewer mixing steps • Does not require heating • Gives a more stable colorimetric response than the assays described above • Its response is prone to influence from non protein sources, particularly detergents • Becomes progressively more nonlinear at the high end of its useful protein concentration range.  The response is also protein dependent, and varies with the composition of the protein. (See examples)   These limitations make  protein standard solutions necessary.

  6. Bradford Metanol Dye stock - • CoomassieBlue G (C.I.# 42655) (100 mg) is dissolved in 50 mL of methanol. • (If turbid, the solution is treated with Norit (100 mg) and filtered through a glass-fiber filter.)  • The solution is added to 100 mL of 85% H3PO4, and diluted to 200 mL with water.  • The solution should be dark red, and have a pH of -0.01. • The final reagent concentrations are 0.5 mg/mL Coomassie Blue G, 25% methanol, and 42.5% H 3PO4.  The solution is stable indefinitely in a dark bottle at 4°C.

  7. Bradford – Etanol 95 • Bradford reagent: • Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, • add 100 ml 85% (w/v) phosphoric acid. • Dilute to 1 liter when the dye has completely dissolved, and • filter through Whatman #1 paper just before use. • (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).

  8. Preparación del Bradford

  9. Ensayo • Assay reagent - • diluting 1 volume of the dye stock with 4 volumes of distilled H2O.  The solution should appear brown, and have a pH of 1.1.  It is stable for weeks in a dark bottle at 4°C. • Protein Standards - Protein standards should be prepared in the same buffer as the samples to be assayed.  A convenient standard curve can be made using bovine serum albumin (BSA) with concentrations of • 0, 250, 500, 1000, 1500, 2000 µg/mL for the standard assay • 0, 10, 20, 30, 40, 50 µg/mL for the microassay.

  10. Protocolo • Rotular 8 cuvetas de espectrofotometría del 1 al 7 y la octava como B (Blanco) • Servir 1ml de la dilución de BrPD en las 8 cuvetas de espectrofotometría para la curva de calibración. • Servir volumen equivalente a 0ug, 2ug, 4ug, 6ug, 8ug, 12ug, 16ug 24ug de BovineSerumAlbumin (BSA) • Mezclar agitando suavemente entre los dedos. • Incubar a RT por 5 minutos. • Leer en espectrofotómetro a 595nm • Servir 1ug de cada muestra y leerlas con el mismo blanco de la curva de calibración.

  11. ProtocoloContinuacion • Preparar una gráfica con X= ugs de proteína, Y= Abs a 595nm. • Obtener la línea recta que mejor une los puntos. • Obtener la ecuación de la recta. (y= mx +b) • Analizar la lectura de cada muestra y con la misma curva (ecuación) de calibración • Calcular volumen para obtener entre 12, 15 o 20ugs de cada muestra para servir por carril en una PAGE.

  12. Curva BSA

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