Antibody Detection and Western Blot Analysis

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In today's lab we will be using two different antibodies to detect our protein.  The first was made in a chicken against a fairly conserved region of the large subunit of the Rubisco protein. It is from a company in Sweeden called AgriSera.  This anti-Rubisco antibody that is specific for what you are trying to look for is often called the primary antibody.   The second antibody you will use is an antibody that was produced in a goat that binds to all chicken antibodies.  This is called a secondary antibody.  Secondary antibodies are usually chemically modified by linking them to an enzyme that allows for a colorimetric reaction or luminescent reaction to detect our product.  In the picture below the secondary goat anti-chicken is attached to the enzyme called  alkaline phosphatase.  This enzyme is able to break down a colorless substrate,  to a purple colored substance.  See the picture below.   You can use the amount of color produced to get some idea of how much protein is located in a particular plant.  How could you quantitate the Rubisco signal?
 
 

Picture courtesy of : http://www.vu-wien.ac.at/i123/virokurs/western2.jpg

Thought you should learn some German too :)


Requires:  TTBS, primary antibody, secondary antibody, nonfat dry milk, detection reagent

1.  Because of the time this will take start the first steps immediatly when you come into the classroom. Remove your membranes that you stored in the blocking solution from the refrigerator.  Carefully dump out the blocking solution and wash away excess blocking solution by rinsing one time with TTBS for 15 minutes at room temp with gentle agitation and then for two more times with TTBS for 5 minutes each. Remember to keep your membrane moist at all times. Do not dump out one solution until your ready with the next...

2. During the last wash dilute primary antibody to 1:5000 (recommended 1:2000- 1:20 000) in15 ml of blocking buffer (TTBS with 5% nonfat dry milk). (if using the alternative protocol do not use milk just dilute antibody in TTBS)

3.  Once you have finished washing off the excess blocking agent decant out the TTBS and then wash the membrane in blocking solution containing the primary antibody 1 hour at room temperature with gentle agitation.

4. Decant primary antibody solution. and wash the membrane three times with TTBS with agitation at room temp.  First for 15 minutes, and two more times for 5 minutes each.  This allows you to get rid of any primary antibody that did not stick appropriatly to the membrane.

5. During the washing step dilute the secondary antibody bovine anti-chicken alkaline phosphatase to 1:12000 with 15 ml TTBS containing 5% nonfat dry milk. (If using the alternative reaction use a 1:15,000 dilution of secondary goat anti chicken antibody linked to the enzyme horseradish peroxidase and do not use milk during the incubation of the secondary antibody only TTBS)

6. Incubate the membrane with agitation in the diluted second antibody for 1 hr at room temp.

7. Wash 1 X 15 minutes and 4X5 minutes in fresh changes of TTBS. Procede to appropriate steps below.

For Color Reaction

8. During the last wash make up the detection reagent. To 15 ml of 1x AP color development buffer add 150 ul of AP color reagent A (contains nitroblue tetrazolium, This is toxic- use caution!) and 150 ul of AP color reagent B ( contains 5-bromo-4-chlor-3-indolyl) and mix well.

9. Remove the TTBS from the gel and immerse the membrane in the color development solution and incubate until color development is complete.

10.  Stop the development by washing the membrane in dd water for 10 min. to remove residual color development solution.

11. Dry the membrane. Take a digital photo of your blot to use on your web page.

Alternative Procedure for light reaction

8. During the last wash in TTBS make up staining solution using 6 ml of luminol/enhancer and 6 ml of peroxide buffer.

9. Incubate blot in mixture for 5 min. During this time make sure blot is completely covered and does not dry out.

10. After 5 min. Remove blot from buffer and touch edge to a paper towel to remove excess liquid but do not allow blot to dry.

11. Cover blot with saran wrap and expose to xray film from 30 sec to 2 min or use the BioRAD Chemilumenescent camerea to take picture. Exposure time may take longer than 2 min., this is just a starting point.


tkr --Oct. 1, 2005