Presentation on theme: "Fundamental Of Bioprocess Engineering 1 Dot Blotting Ch-ng Tau Poppy Yang."— Presentation transcript:
Fundamental Of Bioprocess Engineering 1 Dot Blotting Ch-ng Tau Poppy Yang
What Is Dot Blotting It is introduced in 1970s, to identify antigens that bound to specific antibodies It can be used either as a qualitative method for rapid screening of large number of samples or as a quantitative technique Many different way to do dot blotting, e.g Electroblotting Many detection methods e.g.Radioactive We are using the simplest and cheapest way --manual spotting qualitative method
Theoretical Background Aim of the experiment: identify ovalbumin among casein and gluten Objective of the experiment : produce a stain on the nitrocellulose membrane Proteins are complicated amino acid sequence, have high molecular weights, classified according to biological function, source and occurrence Antibody are amino acid sequence too, can attach to the specific protein
Theoretical Background P1P3P2 P1 PA P3 PA P2 PA Add PA Add developing solution Add SA P1 SPA P3 SPA P2 SPA
Theoretical Background Developing solution is added to produce stain: in the developing solution, in the presence of hydrogen peroxide, dye is oxidised and form an insoluble white coloured compound on the ovalbumin dot As secondary antibody occupy the whole membrane, developing solution make the membrane to blue colour.
In This Experiment Proteins used:casein, ovalbumin, and gluten----10ml, 0.1 %(w/v) in urea Blocking solution: BSA (Bovine Serum albumin)---250ml, 2 %(w/v) in Tris-Cl Primary antibody:anti chicken egg (ovalbumin) developed in rabbit---12ml, 0.01%(w/v) in Tris-Cl
In This Experiment Secondary antibody:anti rabbit IgG developed in goat---12ml, 0.01 %(w/v) in Tris-Cl Developing solution:100ml, contains 0.48mM 4-Chloro –naphthol, 17%(v/v) methanol, and need to add H 2 O 2 at concentration of 0.01%(v/v) just before we need to use developing solution
Procedure Cut 1×8cm nitrocellulose paper strips Prepare solutions needed in the experiment Concentration of antibody solutions can be much smaller than protein solutions, as the reaction is very sensitive, and we can save some of the expensive antibodies
Procedure Eject 2µL each for the three different protein solutions along the strips IN AN ORDER! Or you may be very confused when facing the result! Be very careful to avoid contamination of protein sample! COG
Procedure Dry the dots Soak in primary antibody Soak in Tris-Cl Soak in blocking solution Soak in secondary antibody Soak in Tris-Cl 10min 5min 1hour5min 1hour 5min What we need to do in this 5min waiting time?….see next slide please!
Procedure Prepare the developing solution quickly and place the strip into it immediately Look it up after 10min to find out if there is any colour developed The expect result is: COG
How Did We Do The Experiment We actually did not prepare the solutions we need in exact concentration We did not soak the strip in the solutions as long as it suppose to be as shown in the procedure During the experiment, we make a lot mistakes( say scratch the strip), which may affect the result
Experiment Result We did not get the expected result! the gluten’s dot show blue—good! However, the casein dot suppose to be blue as well—negative result! COG
Discussion Casein may not blocked to the strip, or may be blocked but got scratched off? Antibody did not occupy the casein dot? Perhaps when the strips were being dried, casein deposited out as the solid, and show the colour in the later steps? casein was contaminated by ovalbumin?
Further Discussion Be more accurate and careful when doing the experiment. Leave the strip in all the solutions longer. Do further research to improve the experiment (say use a different way or solution?) Quantitative test to the strip can be applied, as a extension to the qualitative method that we have so far