BMP: EXP1 Protein determination

Question 1

What is a UV spectrum?

Answer 1

• It is a ranage of wavelengths part of the EM spectrum just above visable light. 10nm - 400nm.
• Absorbance is proportional to the number of molecules in the light path (Beer Lamber Law)

Question 2

What is Wavelength max?

Answer 2

THe wavelength at which the maximum fraction of light is absrobed by a solution

Question 3

What were the wavelength max for the Biuret and Lowry determinatinos?

Answer 3

• Biuret - 540nm
• Lowry - 600nm

Question 4

What is Beer LAmberts Law?

Answer 4

A=Σlc

• A = Absorbance
• E = molar extinction coeficent (The measure of how strongly a chemical absorbs light at a specific wavelength
• l = path length of light
• c = concentration in mol/L

Question 5

When performing a quantative analysis for protein concentration what first needs to be done?

Answer 5

The determination of linear range otherwise may be errors

Question 6

What can be said about proteins and Absorbance relationship?

Answer 6

Dyes bind to proteins non-covalently but tightly.

At high concentrations the relationship between proteins and absorbance isn’t always linear

Question 7

What is the difference between accuracy and presion?

Answer 7

Accuracy - values close to correct results

Precision - values all close together

Question 8

What are systematic errors?

give examples

Answer 8

Errors which cause the results to be in error in the same sense e.g. all too high/ low. Causes results to be inaccurate

• Failure to work in linear range
• Waterbath - even tho on dile is 37 may not be
• No calibration of pipettes/ UV spec

Question 9

What are random errors?

Answer 9

Errors which cause results to fall on both sides of the average value. Expressed in 3 ways:

• Repeatability: spread of replicate determinations measured under a defined set of contditions
• Intermeidate precision: expresses laboratory variation of precision when carried out by different [eople on different days
• Reproducibitity: measure of between laboraty precisoin

Question 10

What does %RSD tell you? What is the aim?

Answer 10

A measur eof precision of results. <5%

Question 11

What does a spectrophotometre do?

Answer 11

Measures absorbance of light by substances in a solution

Question 12

What is the absorption of light depenent on?

Answer 12

• Absorption of light directly proportional to the number of absorbing solute molecules encountered
• Absorption of light is exponetially related to the path length of light

Io/I = Ecl

Io = intesnity of incident light

I - intensity of transmitted light

Question 13

Describe what happens during the biuret assay?

Answer 13

The biuret reaction involves a reagent containing copper (cupric) in an alkaline solution. Molecules containing 2 or more peptide bonds associate with the cupric ions to form a coordination complex, by reducing the copper ions from cupric to cuprous form. This produces a faint blue-violet colour and Amax = 540nm.

The copper is reduced and electrons released

Question 14

What agent is used to help prepare a standard calibation curve?

What is its sensitivity values?

Answer 14

Bovine albumin serum (BSA)

1-10mg ml-1

Question 15

Describe the steps involved in performing the biuret assay

Answer 15

1. Prepare test tubes with set amount of BSA and water to 2ml.
2. Add 3ml of biuret reagent to each and mix by inversion. Incubate in waterbath at 37C for 30 mins before allowing to cool and measuring Absorbance
3. Construct a calibration graph and determine unknown conc from Absrobacne measured

Question 16

Describe the mechanism behind the Lowry method

Answer 16

BAsed on the reactoin between certain AA in a protein with sodium tungtate and sodium molybdate in phosphoric acid the active constituents in folin-ciocateau phenol reagent.

Tyrosine, tryptophan and to a lesser extent cystine, cyseine and histidine react.

The AA reduce the mixed metalloacids by causing the loss of 1 to 3 oxygen atoms from tungastate and/or molybdate. Thereby producing one or more of sveral possible compounds with a characterisitc blue colour. Due to reaction of Cu2+ wth peptide bonds and reduction of a complex reagent containing phosphomolybdate.

Question 17

What is the sensitivity of the lowry method?

Answer 17

10 -200 micrograms ml-1

Question 18

Describe the steps involved in the Lowry method

Answer 18

1. Prepare test tubes with BSA and water up to 0.5ml
2. Add 5ml of solution C, mix and leave at RT for 10mins
3. Add 0.5ml of Folin-ciocalteau phenol regagent and mix immeditely. LEave for 30mins at RT
4. Measure absorbance at 600nm
5. Plot calibration graph and determine unknown

Question 19

What are the values of tryosine and tryptophan in proteins? What Wavelengths do these absorb?

What are the positives and negatives of the Lowry method?

Answer 19

• 3.5% tyrosine and 1.1% tryptophan
• Absorb UV at 270-280nm
• only semi-wuantative analysis due to protein to protein variations in AA%
• Interferencd caused by contaiminating nucleic acids - they absorb more stongly at 260nm.
• Fast, sensitive and easy
  *

Question 20

Dexcribe the steps in UV absorption

Answer 20

• MEasure Absorbance of tyrosine and tryptophan samples between 200-300nm
• Measure absorbance at 260nm and 280nm or a solution containing protein and nucleic acid.
• USe equation:

Protein mg/cm-3 = 1.45A₂₈₀ - 0.74A₂₆₀

to detemrine protein conc (mg/ml)