revison

Homework 6: SOP revision

Change in SOPs is part of manufacturer’s attempts for continuous improvement of a process or product. PAGE gels can be cast by an analyst (video 1 – mostly FYI), but a faster, more reproducible way is to buy pre-cast gels (used in video 2). In this exercise, you, the QC analyst, are preparing a draft of a proposed revision of your current SOP for detecting a protein product, to an improved one that uses pre-cast gels.

Pick a name for your biotech company. For your revised cGMP compliant SOP, combine the instructions from video 2 for SDS-PAGE, with the instructions for Western blotting given below in text. Video 3 is provided to help you see and understand the Western steps. You will have to stop at step 9 (or 8) of video 2 and continue on to the appropriate Western Blotting step in the provided Western Blot protocol. Note that the Western Blot protocol below is missing parts of what will be considered a cGMP-compliant SOP format – you will have to add those missing parts. You can follow the content and format of an SOP in the “Label control SOP” and in the “SOP Stability Cougar”. Also look for the parts of a cGMP-compliant SOP from lecture ppt 05 on quality documentation. You will have to come up with your own reason/objective and your source of recombinant protein that you will be detecting for your Western. The protein that you are detecting can be from a bacteria, yeast, any plant, any animal, human, etc. You are all expected to work on different proteins. Also do a google search for a vendor/source of antibodies to detect your protein. 4-5pages.

Video 1:

https://www.youtube.com/watch?v=EDi_n_0NiF4

Video 2:

https://www.youtube.com/watch?v=eaETFKXtNRA

Video 3:

https://www.youtube.com/watch?v=VgAuZ6dBOfs

Western Blotting

Materials

Transfer Buffer

750 ml of H2O

100 ml of 10X SDS-PAGE buffer

150 ml of MeOH

PBS-Tween

895 ml of H2O

100 ml of 10X PBS

5 ml of 10% Tween

Blocking Buffer

50 ml of PBS-Tween

2.5 g of Carnation Dry Milk

Dry Gel Solution

385 ml of H2O

200 ml of MeOH

30 ml of 50% glycerol

1. Run protein gel @ 120 – 160 mV.

Standard Marker: 2 l of Perfect Protein Western Marker (Novagen #69959)

or ECL protein molecular weight markers (Amersham # RPN2107)

2. Set up for blotting

1) Cut out a membrane (Immobilon-P) square that will fit gel.

2) Soak membrane in MeOH for 1 min. and then immerse in H2O.

3) Place the membrane in transfer buffer for more than 15 min.

4) Put the blotting gel box in the container filled with transfer buffer, add two sponges to the box, and then place a piece of filter paper onto the sponges (Make sure both are completely wet in the transfer buffer).

5) Remove the gel from running apparatus using spatula with sharp edge.

6) Slice a bit of the gel lane edges and place the gel onto the filter paper on the box (Make sure no air bubbles are between gel and paper!).

7) Place the soaked membrane (from step 3) onto the gel, again making sure there are no air bubbles between the gel and membrane.

8) Place a filter paper onto the membrane, making sure no air bubbles are between paper and membrane.

9) Place two sponges and lid, put these blotting sandwich into the gel box.

10) Fill the box entirely with transfer buffer (If the box is leaking, the apparatus is not assembled correctly).

11) For transferring, run the blot at 30 volts for 2.5 hours.

3. Binding the antibody

1) Slice the membrane around the gel using a razor blade in order to minimize area of membrane that is to be probed with antibodies and stained, when protein is finished transferring.

2) Incubate the membrane in dry milk blocking buffer with shaking for one hour at room temperature or overnight at 4C.

3) Put 2 l of S-Protein HRP conjugate (Novagen #69047) and 5 l of anti-FLAG M2 Monoclonal Antibody (Sigma # F3165) into 10 ml of SuperBlockBlocking Buffer in PBS (PIERCE #37515).

4) Place the membrane in heat sealable bag and pour this solution, seal the bag (be sure to cool seals of bag down) and incubate for one hour for nutating.

5) Wash membrane 3 times for 15 minutes each with PBS-Tween on shaker.

6) Mix 2 l of Anti-mouse Ig-HRP linked whole antibody (from Sheep) (Amersham #NA931) and 2 l of S-Protein HRP conjugate with 10 ml of dry milk blocking buffer.

7) Place membrane in heat-sealable bag and add the antibody solution. Seal bag, being careful not to allow antibody solution to touch recently sealed edges.

8) Incubate one hour on shaker.

4. Developing the blot

1) Wash membrane 3 times for 15 minutes each with PBS-Tween on shaker.

2) Mix 4 ml of solution A with 100 l of solution B (ECL Plus Westernblotting Detection Reagent, Amersham # RPN2132).

3) Place membrane on a piece of parafilm (protein side up), add the staining solution to the membrane and gently tilt it to ensure protein is covered in solution.

4) Cover the membrane in foil and incubate for 4 minutes.

5) Wrap membrane in plastic bag and place it in the film box.

6) Take this to the dark room and burn the film for 5 minutes, or as much time as would give a good signal.

7) Slide the film through the developer to develop.