Topic%2004%20_%20Protein%20Assays%20_%20F10

Topic%2004%20_%20Protein%20Assays%20_%20F10 - Protein...

Info iconThis preview shows pages 1–10. Sign up to view the full content.

View Full Document Right Arrow Icon
Protein Assays heavily used technique in life sciences many protein assays Lowry Peterson Silver Ponceau S Biuret Amido Black NanoOrange A660-Assay BCA ← common Bradford A280 ← common & Exp 2
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Protein Assays Choosing protein assays: Assay Property Avoid BCA reduction of Cu ++ to Cu + redox agents Cu + bind to BCA reagent Bradford hydrophobic dye detergents, lipids, binding membrane proteins A280 absorption of UV light conjugated double by aromatic rings bonds ( π e - systems)
Background image of page 2
Protein Assays Basic idea: Abs unknown = Abs ∆ Abs = slope of plot [?] unknown [standard] ∆ [standard] Abs vs [standard]
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
A corr = (A – blank) of the tube with unknown [protein] protein concentration ( eg mg/ml) of unknown [protein] Concentration of standard protein: BSA A corr Abs vs [BSA] : standard curve
Background image of page 4
A 280 (UV) Assay 1. direct assay, not a derivative assay 2. presence of trp, tyr, phe 3. range: 0 to 1 mg/ml (not very sensitive) 4. advantage: non-destructive
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
A280 assay: calculating the unknown protein concentration Measure Abs for a dilution series of the protein solution don’t know the [protein] need at least one dilution to fall in range of the standard curve [protein] undiluted = ([X μ g/ μ l])(dilution factor) from standard curve or calculated from slope dilution factor = inverse of the dilution
Background image of page 6
Bradford assay: Coomassie Blue G-250 1. complex binding characteristics in general binds to hydrophobic surfaces, regions 2. standard curve typically a second-order polynomial 3. range ~0.002 μ g/ μ l to ~0.024 μ g/ μ l 4. disadvantage: destructive to proteins
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Overlay spectra: Bradford reagent ± protein 500 600 λ nm A Bradford reagent only A 465 A 595 increases with protein Bradford reagent + protein
Background image of page 8
Here’s what the overlay really looks like: apply the concepts from previous slide
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 10
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 11/21/2010 for the course MCB 120L 69059 taught by Professor Fairclough during the Fall '10 term at UC Davis.

Page1 / 19

Topic%2004%20_%20Protein%20Assays%20_%20F10 - Protein...

This preview shows document pages 1 - 10. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online