20.109(S07): Western analysis

Introduction

"Divide and conquer" may be an effective military strategy but its usefulness is not limited to that arena. The reductionist approach has been an important means of understanding complex biological processes. By tweezing apart networks and pathways, the components that contribute to the overall behavior of the system may be understood in great detail. As you've seen, however, reassembly of the component level understanding into a predictive and quantitative models for the system isn't always straightforward. That's what happened with the T7 model that you read about last time, and their response to the limited success of the models is what makes that T7 work so novel. Rather than continue to tweeze apart and better understand the natural example, they built a surrogate T7 that was a better template for experimental work, easier to manipulate and analyze, easier to characterize and understand.

Over the last few weeks you have gained important and detailed understanding of the natural M13 bacteriophage, and through your molecular manipulations to epitope-tag one of two phage proteins, you are both testing the existing knowledge of the system and extending it. Today you will see if the manipulation you have carried out is tolerated by the phage, specifically, if the tagged protein is detectable in bacteria that are infected by your manipulated phage and if the phage life cycle is altered. These will be determined through Western analysis, looking for proteins that react to an anti-myc antibody, and by plaque assay, with which you are already familiar.

You will also help design a surrogate M13. A rough sketch of this genome is included as Part 3 of today's lab and while your protein gel is running, you and your lab partner should examine the draft and refine it. Based on everyone's design ideas, we will compile the surrogate M13 to test later in the term.

Protocol

Part 1: SDS-PAGE

Two groups will share one acrylamide gel.

1. Retrieve the myc-tagged candidate from last time.

SDS-PAGE AND TRANSFER

Two groups will share one acrylamide gel.

1. Retrieve the eGFP-1, siRNA ctrl-1, and oligofect-1 samples that you froze last time. Calculate the volume of Sample Buffer needed to resuspend the cells at a concentration of 1 x 103 cells/ul. Sample Buffer contains glycerol to help your samples sink into the wells of the gel, SDS to coat amino acids with negative charge, BME to reduce disulfide bonds, and bromophenol blue to track the migration of the smallest proteins through the gel.

1. Put on gloves then add the calculated volume of Sample Buffer to your cells. Boil the eppendorf tubes with lid locks for 5 minutes. Spin your samples in the microfuge 1 minute to pellet your cells.

1. Put on gloves. Load the indicated volumes of each sample onto your acrylamide gel in the order below. Once you have loaded a sample from one tube, move it to a different row in your eppendorf rack. This will help you keep track of which samples you have loaded.

Lane Sample Load 1 Molecular Weight Standards 10 ul 2 eGFP siRNA 15 ul 3 siRNA control 15 ul 4 oligofectamine control 15 ul 5 Leave this lane empty xxx 6 Molecular Weight Standards 10 ul 7 eGFP siRNA 15 ul 8 siRNA control 15 ul 9 oligofectamine control 15 ul

1. Once all the samples are loaded, turn on the power and run the gel at 200 V. The molecular weight standards are pre-stained and will separate as the gel runs. The gel should take approximately one hour to run. During that hour, you should work on your siRNA design for silencing ATM, ATR or Exo1 (see below).

1. Wearing gloves, disassemble the electrophoresis chamber.

1. Blot the gel to nitrocellulose as follows:

• Place the gray side of the transfer cassette in a tupperware container which is half full of transfer buffer. The transfer cassette is color-coded so the gray side should end up facing the cathode (black electrode) and the clear side facing the anode (red).
• Place a ScotchBrite pad on the gray side of the cassette.
• Place 2 pieces of 3 mm paper on top of the ScotchBrite pad.
• Place your gel on top of the 3 mm paper.
• Place a piece of nitrocellulose filter on top of the gel. The nitrocellulose filter is white and can be found between the blue protective paper sheets. Wear gloves when handling the nitrocellulose to avoid transferring proteins from your fingers to the filter. Gently press out any air bubbles caught between the gel and the nitrocellulose.
• Place 2 more pieces of 3 mm paper on top of the nitrocellulose.
• Place a second ScotchBrite pad on top of the 3 mm paper.
• Close the cassette then push the clasp down and slide it along the top to hold it shut.
• Place the transfer cassette into the blotting tank so that the clear side faces the red pole and the gray side faces the black pole.

1. Two blots can be run in each tank. When both are in place, insert the ice compartment into the tank. Fill the tank with buffer. Be sure the stir bar is able to circulate the buffer. Connect the power supply and transfer at 100 V for one hour. You can use this time to complete your siRNA design or read the attached article for next time.

1. After an hour, turn off the current, disconnect the tank from the power supply and remove the holders. Retrieve the nitrocellulose filter and confirm that the pre-stained markers have transferred from the gel to the blot. Stain the blot with Ponceau S to confirm that the lanes were equally loaded and transferred. Finally, move the blot to blocking buffer (TBS-T +5% milk) and store it in the refrigerator until next time.

Protocols

Part 1: Protein gel

1. run acrylamide gel
2. blot

Part 2: Plate candidate sup to look for phage

1. plate sup for plaques

Part 3: M13 renovation

1. refactoring details...

DONE!

Reagents list

• Transfer Buffer
  • 25 mM Tris
  • 192 mM glycine
  • 20% v/v methanol