Full name: …………………………………………………… Student ID.: ……………………
Prac day:
Demonstrator: ……………………………………Bench number: …………
This worksheet is due to be handed in to your demonstrator for marking at the start of prac class in week 5
BCH2MBC – 2017
PRAC UNIT 2
PURIFICATION & CHARACTERIZATION OF IgY FROM CHICKEN EGG YOLK
WORKSHEET #2
for Sessions 2 and 3
Part 1 -Some theoretical aspects of SDS-PAGE
Question 1:
Why are the following reagents added to the gel, the sample loading buffer and/or the electrode buffer during the SDS-PAGE procedure? What is the purpose of each of these compounds?
(i) the anionic detergent sodium dodecyl sulphate (SDS):
(3 marks)
(ii) glycerol:
(1 mark)
(iii) a reducing agent such as dithiothreitol(DTT) or beta-mercaptoethanol(ßME):
(2 marks)
(iv) the blue-coloured dye bromophenol blue:
(2 marks)
Question 2:
Fill in the blanks in the following statement:
“Under the conditions used during SDS-PAGE, all proteins carry a large ……………… charge. This causes them to move toward the ………………electrode which is called the ……………… . Because all proteins now carry the same charge density and are fully linearized, their separation is based solely on their ……………………… .”
(4 x ½ = 2 marks)
Question 3:
The SDS-PAGE separative procedure employs a ‘discontinuous’ system in which proteins in the sample initially pass through an upper ‘stacking’ gel before they enter the lower ‘separating’ gel in which they are separated on the basis of size.
(i) What is the purpose of the ‘stacking’ gel?
(1 mark)
(ii) What problem may arise if such a system is not used? If you attempt to run SDS-PAGE without a stacking gel, what would be the likely effect on the resolving power of the system (i.e. its ability to physically separate different proteins into visibly distinct bands on the gel)? Explain your reasoning.
(2 marks)
Question 4:
After SDS-PAGE, the gel is stained with Coomassie Blue to visualize the proteins. Coomassie Blue binds to proteins via electrostatic (ionic) interactions by virtue of its fairly extreme pKa value.
(i) The gel staining solution contains 10% (v/v) acetic acid, making it quite acidic with a pH of approx. 2.5. What is the sign of the charge on almost all proteins at pH 2.5?[Note: the charge previously furnished by SDS is no longer relevant at the gel staining stage]
□ negative □ zero □ positive
(ii) To form electrostatic interactions with proteins at pH2.5, what must the sign of the charge on Coomassie Blue be at pH2.5?
□ negative □ zero □ positive
(ii) What conclusion can you draw about the pKa of Coomassie Blue?
□ must be below 2.5 □ must be exactly 2.5 □ must be above 2.5
(3 x ½ mark = 1½ marks)
Part 2 -Qualitative analysis of your IgY purification samples by SDS-PAGE
Question 5:
View samples S1 and P1, the supernatant and pellet resulting from yolk treatment with 4% PEG.
(i) Briefly comment on the complexity of these samples from this early stage of the purification, i.e. discuss the number of proteins present in comparison with later samples.
(1 mark)
(ii) In which fraction should the IgYhave been at this stage (tick one of the boxes below)?
Can you see any IgY in these samples (compare with your final IgY preparation, sample P3). If so, is it in the correct fraction? What may stop you from seeing any IgY at all in these samples from this early stage of the purification?
IgY should be in: □ supernatant S1 or □ pellet P1
(2 marks)
Question 6:
View samples S2 and P2, the supernatant and pellet resulting from treatment of the previous supernatant S1with 12% PEG.
(i) In which fraction should the IgY have been at this stage (tick one of the boxes below)?
Can you see any IgY in these samples (again, compare with your final IgY preparation, sample P3). If so, is it in the correct fraction at this stage?
IgY should be in: □ supernatant S2 or □ pellet P2
(2 marks)
(ii) Briefly discuss the purity of the IgY at this intermediate stage of the purification. Comment on the number of proteins present in comparison with the earlier S1 and P1 samples.
(2 marks)
Question 7:
View samples S3 and P3, the supernatant and pellet resulting from treatment of the previous pellet P2 with 12% PEG for a second time. This second 12% PEG treatment essentially ‘washed’ the IgY from pellet P2 to give the final IgY preparation.
(i) In which fraction should the IgY have been at this stage (tick one of the boxes below)?
Is your IgYvisiblein the correct fraction?
IgY should be in: □ supernatant S3 or □ pellet P3
(1 mark)
(ii) Discuss the purity of the IgY at this final stage of the procedure. Comment on the number and relative amounts of the protein bands observed. Was your IgY purified to homogeneity (100% pure)? Give a rough visual estimate of the % purity achieved.
(3 marks)
Question 8:
All six samples analysed so far were run on SDS-PAGE under reducing conditions, i.e. in the presence of dithiothreitol (DTT). The seventh samplecontained an aliquot of your final IgY preparation (P3, similar to the sixth sample) except that it was applied to the gel under non-reducing conditions, i.e. in the absence of a reducing agent. Describe what you see in this seventh sample, and explain whether the result is consistent with your knowledge of the structure of IgY.
(3 marks)
Part 3 -Determination of the MWs of the light and heavy chains of IgY
The molecular weight (MW) or size of a protein can be measured experimentally in a number of ways. Increasingly, the technique of mass spectroscopy is used to measure molecular masses with amazing accuracy (to within 1 Da) but it is best applied to highly purified proteins and requires very expensive instrumentation. SDS-PAGE can also be used to determine the size of proteins, and although it is far less accurate than mass spec, it is adequate for many applications and is routinely used in many laboratories because it doesn’t require particularly sophisticated or expensive equipment.
There is an inverse logarithmic relationship between MW and the migration rate of proteins on SDS-PAGE, so a graph of log10MW versus migration rate (or distance moved) will give a straight line with a negative slope.
The MW markers applied to your gel will be used to generate a MW calibration curve, then the distances moved by the polypeptide components of your purified IgYsample (sample P3,applied to the gel under reducing conditions) will be used to determine the MWs of the light (L) and heavy (H) polypeptide chains of IgY by interpolation on the graph.
Adapted from Lehninger Principles of Biochemistry (used with permission)
Before attempting to estimate the sizes of the L and H chains of IgY, note that they appear asrelatively diffuse bands, i.e. they each cover a range of MWs (rather than the highly-resolved bands typically observed for other proteins such as the MW markers in lane 8). This is because:
(i) Antibodies undergo a post-translational modification called ‘glycosylation’ involving the addition of sugars (oligosaccharides) to form ‘glycoproteins’. This was discussed briefly in lectures in 1st semester and will be elaborated on when the protein secretory pathway is discussed in 2nd semester lectures. Glycosylation is crucial for protein targeting, i.e. directing proteins to their correct destinations or sites of action. Antibodies are synthesized in B-lymphocytes, disulphide-linked and glycosylated, then directed through the secretory pathway to be exported out of those cells for use elsewhere (eg. in serum or egg yolk). The number and types of linked sugars vary,so these antibodies exhibit a range of sizes.
(ii) IgYfrom egg yolks (just like antibodies collected from serum) is termed ‘polyclonal’, i.e. it is a mixed population of antibodies generated against not only a broad range of antigens (foreign proteins), but also against multiple ‘epitopes’ (structural features) on the surface of each of those proteins. Antibodies directed against each antigen possess different amino acid sequences in the variable regions of their light and heavy chains, and this also contributes to the range of sizes observed.
Question 9:
Use the table and other information provided on your SDS-PAGE result sheet (available on the LMS a few days after your second prac session) to complete the following steps:
1. The identities and sizes of the protein MW markers are provided in the table on your results sheet. Calculate the log10 of the MW for each of the MW markers and enter your results into the table.
(½mark)
2. Identify the bands corresponding to each of the MW markers in the centre lane (lane 8) of your gel. To help identify the pre-stained MW markers correctly, some are coloured differently to make them easier to assign – most are stained blue, but two of the markers are mauve or crimson red in colour (see the table on the results sheet for details).
3. Measure the distance travelled by each of the MW markers, i.e. the distance moved from the start of the separating gel (near the top of the image) to the centre of the protein band. Remember that you are measuring the distances moved, so you must measure from the top down, not from the bottom up. For accuracy, measure to the nearest 0.5mm or so, and enter your results into the table.
(1 mark)
4. Prepare a MW calibration curve of log10MW (y-axis) versus distance moved in mm (x-axis). Take note of the following points, and see Steve Jones for more details if necessary:
a. If generating your graph manually (on paper), make it as large as possible (i.e. fill the entire page) to ensure the most accurate interpolated results later on.
b. For the same reason, choose your axis scales and ranges carefully so that the data points fill the entire graph area (as little empty space as possible) – do NOT extend the x and yaxes to zero, as doing so will reduce the accuracy of your interpolations.
c. Although the relationship between log10MW and mobility is theoretically linear, in practice you may see a noticeable curve in the graphwhen such a broad range of MWs is being covered. If this is the case, fit a smooth curve through the data points (i.e. don’t necessarily force a straight line through all of the points).
(4 marks)
5. In the lane containing your final IgY preparation under reducing conditions (may be either lane 6 or 10), measure the distance moved by the light and heavy chains and enter the results into the table on your results sheet. Remember that due to the diffuse nature of these immunoglobulin chains on the gel, you will need to measure distances to the lower and upper extremes of each chain band on the gel – this will enable you in the steps below to determine a range of MWs (i.e. minimum and maximum values, rather than a single value) for each chain.
(1 mark)
6. Estimate the log10 of the minimum and maximum MWs of each chain by interpolation on your calibration curve and enter the results into the table on your results sheet.
(2 marks)
7. Use these values to calculate the range of MWs (lower and upper limits) and an average MW value for the light and heavy chains of IgY, and enter your values into the table.
(1 mark)
8. How do your experimentally-determined estimates of the MWs of the light and heavy chains of IgY compare with literature values? Briefly discuss your findings.
(2 marks)
Remember to attach your SDS-PAGE results sheet (with the table completed) and your MW calibration curve to this worksheet for marking purposes.
Total marks available for worksheet = 40 marks
Raw mark for Prac Unit 2 Worksheet #2 / 40
Deduction for lateness (5%, or 2 marks out of 40, per day or part thereof) / 40
Adjusted mark for worksheet #2 (after deduction for lateness, if any) / 40
Lab performance mark for SDS-PAGE work (session 2, week 7 or 8) / 5
Mark based on quality of SDS-PAGE results / 5
Overall mark for SDS-PAGE work (session 2 plus approx. half session 3) / 50
Final mark (approx. 1½ sessions, so converted to a mark out of 15) / 15
BCH2MBC 2017 Prac Unit 2IgY – Worksheet 2 (for sessions 2 and 3).docx