Bradford Assay

Bradford Protein Assay Practical Report 1. Present your data (including raw data and calculated concentrations) for the protein standards in the form of a clear table. Give one example of how you calculated protein concentration. Do not forget a descriptive title and units (4marks) Title either too long or not descriptive or absent Your results are in duplicate shouldn’t be referred to as ‘set1 set2’ or ‘original’ and ‘duplicate’ Many of you think units of absorbance are nm but A has arbitrary (ie no) units. nm indicates the ax of the chromophore Failure to give correct units in legends eg (ml) or (? g/ml) 2. Plot a graph of absorbance against protein concentration by hand. The graph should have an appropriate title and clearly labelled axes. Staple graph to the completed proforma and the Life Sciences submission sheet (4 marks) Mainly ok but both duplicate Abs- blank should be plotted and one line of best fit drawn through points. Do not extrapolate beyond the highest standard, you have no evidence that Beer-Lambert’s Law applies at high A. Make sure you choose appropriate scale and use full scale deflection on A4 graph paper. These types of graph are standard curves and that term should be in the title, remember we are not directly measuring the absorbance of protein, but a chromophore derived from the protein. 3. Present your data for unknown samples (including raw data and calculated concentrations of X Y) in the form of a clear table. Do not forget title and units. (4 marks) All data should be in one table but pay attention to typesetting and make sure that words/numbers are not split between 2 lines, this will lose marks. Absorbance of blank must be subtracted from values for unknown as they also contain non-specific absorbance. Many of you wrote dilutions incorrectly eg 1:2. The symbol : means ratio ? this actually means 1in 3. Either write as 1in 2 or 1:1 Never average absorbance-it’s not good practise (except for blank) you should convert to analyte then average your final results. Most dilute samples have least absorbance, many of you muddled your dilutions making final values incorrect. Always double-check arithmetic. If the final answer for the different dilutions don’t agree, look at your results and ask yourself if they seem right. Remember there is only one right answer for each unknown . Explain briefly each step of your calculations to find the protein concentration of X and Y, underlining your final answers. Convert to mg/ml. (6 marks) No need to explain how to read values from the std curve. Explain which absorbance values you read from the graph, what (if any) dilution factor you multiplied that value by, and then which answers you then averaged to get your final answers and why you ignored any data (eg poor duplicates or off scale cf standard- you cannot extrapolate beyond your std curve ) Some of you not using the proforma wrote too much. You will be penalised for exceeding allocated space in assignments, so be mindful of this 5. What is the chromophore measured in the Bradford assay? (2 marks) Many of you defined the term chromophore rather than describing the Bradford chromophore which is CBB + protein. (not CBB alone! ) The ? max at 595nm is formed when the dye binds to protein 6. What is the purpose of the blank? Why is it necessary to subtract the absorbance of the blank from all other results? (2 marks) The blank gives us the value for non-specific absorbance ; as we are interested in the specific (in this case protein) absorbance, we must subtract the blank absorbance from all other abs. values. Many of you didn’t subtract the blank from the unknown’s but as they are also mixtures of protein, NaCl and reagents measured in cuvettes, they also contain non-specific absorbance so you must subtract the blank. Many of you said the blank is used to zero the spectrophotometer (which it can be ) but we didn’t do that; we zeroed on water or NaCl then subtracted the blank mathematically. The blank you had to deduct was to remove the combined absorbances of water, NaCl and most importantly the dye in the uncomplexed state 7. The Biuret and Folin-Lowry are two other commonly used colourimetric protein assays. UV absorption can also be used to determine protein concentration. Describe the basis of these methods and compare them with the Bradford assay in terms of ease, sensitivity, range and interferences. (8 marks) You need to describe the biochemical basis(not the actual method) of the Biuret, Lowry and Bradford assays. The Lowry is a modification of the Biuret to improve it’s sensitivity so it’s appropriate to describe the Biuret method first , then describe the Lowry modification You need to state the range (the lowest- highest concentration they can detect) sensitivity(the lowest amount they can detect) for each assay. Some of you confused sensitivity with interference ie substances which, if present will give incorrect results. You need to state how reliable they are -whether they are prone to interferences. You could mention cost of reagents, ease of procedure Many of you placed too much emphasis on the ? ax of the different chromophores described but this is not really relevant. You need to state the wavelength at which proteins absorb UV radiation and which moieties in proteins absorb in the UV. ie at 280nm(near UV) it’s the aromatic amino acids, some of you also mention A200nm(far UV) at which peptide bonds absorb, although this is of little practical use. Note any inte rferences- remember many things absorb UV radiation Advantages of using UV- it’s non destructive so you can recover your sample for further investigation. Formula which relates UV absorption to protein concentration