Biology

Full name:  ……………………………………………………       Student ID.:  ……………………

 

Prac day:

 

Demonstrator: ……………………………………Bench number:  …………

 

From which chicken type was your IgY purified? (circle one)

Free-range       Barn-housed      Caged

 

Thefirst and second parts of this worksheet #3 cover theoretical aspects of the Western transfer procedure and the probing/detection process, and can be started immediately.  The third part, which will guide you through the analysis of your own Western transfer experiment, cannot be started until you see your Western transfer results at the end of session 4 (week 5) and/or view them on the LMS a few days later.

 

BCH2MBC  –  2017

PRAC UNIT 2

PURIFICATION & CHARACTERIZATION OF IgY FROM CHICKEN EGG YOLK

 

WORKSHEET #3

for Sessions 3 and 4

 

Part 1  -Theoretical aspects of the Western transfer procedure

 

Question 1:

Why is the Western transfer process necessary?  After separating a mixture of proteins by SDS-PAGE, why bother transferring the proteins to a nitrocellulose filter (NCF) at all?  Why not simply probe for the protein(s) of interest directly in the gel?  You should be able to think of four reasons.

1.       2.       (continued on next page) 3.       4.       (4 x 1mark = 4 marks)

 

Question 2:

Why should the transfer of proteins from the gel to the NCF be performed immediately after separating the proteins by SDS-PAGE?  What would happen if the gel was left to stand (eg. for a few hours, or overnight) before starting the transfer procedure?

(2 marks)

 

Question 3:

Ponceau S is a general (non-specific) protein dye that stains all proteins a pink colour, and its staining is reversible (i.e. it readily washes off, leaving the proteins as they were).  After the Western transfer, why did you stain the NCF for proteins?  You should be able to think of two distinct reasons.

(2 marks)

 

 

Part 2  -Theoretical aspects of probing Western transfers with antibody and detecting the positions where antibody has bound

 

Question 4:

 

Regarding the quenching (or ‘blocking’) of the Western transfer NCF with ‘Blotto’ before probing with the primary antibody:

1. What is ‘Blotto’? Which protein is its major constituent?

(1 mark)

1. What does ‘quenching’ mean in the context of the Western transfer, and what was the purpose of this quenching step?

(1 mark)

1. What would you expect to see on the NCF at the end of the experiment if this step was omitted? Why?  Explain your reasoning.

(1 mark)

 

Question 5:

The secondary antibody–enzyme conjugate used to probe your Western transfer can be described as “rabbit anti-chicken IgY – AP conjugate”, where AP is the enzyme alkaline phosphatase covalently linked to the antibody for detection purposes.

 

1. Draw in the space below a diagram showing the basic structure of this secondary antibody–enzyme conjugate reagent. On your diagram, label the following features of the structure:
   • the light and heavy chains of the antibody
   • the F_ab (antigen binding) regions of the antibody
   • the F_c(constant) region of the antibody
   • the enzyme component.

(4x 1 mark = 4 marks)

 

1. To which part of the antibody molecule should the reporter enzyme molecule be attached? Why?  Explain your reasoning.

(1½ marks)

 

1. Are there similar considerations regarding the region of the enzyme molecule to which the antibody molecule should or should not be attached? Again, explain your reasoning.

(1½ marks)

 

Question 6:

You will detect IgY-reactive proteins on your Western transfer NCF using a secondary antibody attached to the enzyme alkaline phosphatase (AP).In the presence of BCIP and NBT, AP catalyses the formation of the indigo-coloured NBT-formazan which is depositedas an insolubleprecipitate on the NCF to indicate the position of the target antigens.ELISA-based techniques can also be set up using AP to measure antigens on microtitre plates, using spectrophotometric methods (i.e. light absorbance) to record the results.  In these spectrophotometric assays, the AP enzyme is reacted with a different substrate, p-nitrophenyl phosphate (PNPP), which is cleaved by AP to produce a yellow-coloured soluble product.Why is it essential to have an insoluble AP product for Western transfers but a solubleAP product for absorbance-based ELISA assays?

(2 x 1½ marks = 3 marks)

 

 

Part 3  -Analysis of your own Western transfer results

 

Question 7:

Why were samples of IgY applied to the gel and transferred to the NCF?  What purpose do these samples serve, and what should they show you?

(2 marks)

 

 

Question 8:

The following questions refer to the staining of your own NCF with Ponceau S immediately after the Western transfer was completed.

 

1. What did you see in the lanes containing the IgY samples after Ponceau S staining? How many protein bands were visible?  Was this consistent with what you expected to see in samples of IgYafter staining with a general protein stain?  Were the sizes of the protein bands observed consistent with your knowledge of the structure of IgY?  (use the MW markers on the NCF to make rough visual estimatesof the band sizes – a MW calibration curve is not necessary)

(4 marks)

 

1. What did you see in the lanes containing the mammalian and bacterial cell extract samples after Ponceau S staining? How many protein bands were visible?  Was this consistent with what you would expect to see in crude cell extracts after staining with a general protein stain?

(2 marks)

 

 

Question 9:

The following questions refer to the signals visible on your own NCF after probing with the primary antibody and secondary antibody-enzyme conjugate and adding the AP-detection reagents.

 

1. What did you see in the lanes containing the IgY samples on your own NCF after AP detection? How many stained bands were visible?  Was this consistent with what you expected to see in samples of IgY after adding the rabbit anti-chicken IgY – AP enzyme conjugate?  What did these results tell you, and why was this so important?

(4 marks)

 

1. What did you see in the lanes containing the mammalian and bacterial cell extract samples on your own NCF after AP detection? Did your own IgY preparation recognize and bind to any of the proteins in these two samples?  If so, how many protein bands were visible?

(1½ marks for each extract x 2 = 3 marks)

 

 

Question 10:

The following question relates to the Western transfer results from the entire class.  Images of all the NCFs from your prac class after AP detection will be posted on the LMS the day after your fourth prac session.  After looking at a representative number of NCFs, probed with IgY purified from all three categories of laying chicken (caged, barn-housed and free-range), can you draw any conclusions about the breadth of IgY antibody responses observed in the three categories of laying chicken?  Discuss possible reasons for the differences observed (if any).

(4 marks)

 

Remember to attach hardcopies of your Western transfer NCF images (both after Ponceau S staining in session 3 and after AP detection in session 4) to this worksheet for marking purposes. You’ll see your own results during your own prac sessions, but the results from the entire class will be posted on the LMS within a few days.

Total marks available for worksheet = 40 marks

 

Raw mark for Prac Unit 2 Worksheet #3	/  40
Deduction for lateness (5%, or 2 marks out of 40, per day or part thereof)	/  40
Adjusted mark for worksheet #3 (after deduction for lateness, if any)	/  40
Lab performance mark for Western tfr(sessions3 & 4, wks9/10 & 11/12)	/    5
Mark based on quality of Western tfr results (Ponceau and AP detection)	/    5
Overall mark for Western tfr work (approx. half session 2 plus session 3)	/  50
Final mark (approx. 1½ sessions, so converted to a mark out of 15)	/  15

 

BCH2MBC 2017 Prac Unit 2IgY–Worksheet 3 (for sessions 3 and 4).docx