20.109(S07): Western analysis

Introduction

image credit: Justin Lo, MIT Course 20

"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 the author's 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 our existing knowledge of the system and extending it. Today you will perform experiments to 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

Each group will run a lane with a positive control (a bacterial strain expressing a myc-tagged protein), a lane of molecular weight markers, a lane with bacterial cells expressing the protein you've myc-tagged, and supernatant from that strain. These will be run in duplicate, so you will have two blots to probe next time.

1. Retrieve the bacterial cultures with your myc-tagged candidates that have been stored at 4°C since last time.
2. Make a 1:10 dilution of your cells and of the myc-positive control (50 ul cells plus 450 ul water) and measure their density at an absorbance of 600 nm using the spectrophotometer. If you do not remember how to use the spectrophotometer, please ask the teaching faculty to help.
3. Calculate the volume of 1X sample buffer needed to resuspend the cells at a concentration of 1 OD 600 in 100 ul of sample buffer. 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. Wear gloves when using sample buffer or your hands will get blue and smelly.
4. Mix 50 ul of the supernatant from your myc-candidate with 50 ul of 2X sample buffer.
5. Boil the eppendorf tubes with lid locks for 5 minutes.
6. 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	Volume to load
1	"Kaleidoscope" protein molecular weight standards	5 ul
2	myc positive control, from teaching faculty	25 ul
3	p8+myc or p3+myc candidate, cells	25 ul
4	p8+myc or p3+myc candidate, supernant	25 ul
5	empty
6	"Kaleidoscope" protein molecular weight standards	5 ul
7	myc positive control, from teaching faculty	25 ul
8	p8+myc or p3+myc candidate, cells	25 ul
9	p8+myc or p3+myc candidate, supernant	25 ul
10	M13K07 phage, from teaching faculty	25 ul

7. 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 parts two and three of today's protocol.
8. Wearing gloves, disassemble the electrophoresis chamber.
9. 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 1 pieces of filter paper on top of the ScotchBrite pad.
• Place your gel on top of the filter 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 but thoroughly press out any air bubbles caught between the gel and the nitrocellulose.
• Place another piece of filter paper on top of the nitrocellulose.
• Place a second ScotchBrite pad on top of the filter 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.
  

10. 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 parts 2 and 3 of today's protocol.
11. 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. Move the blot to blocking buffer (TBS-T +5% milk) and store it in the refrigerator until next time.

Part 2: Plaque assay

You will precipitate the phage that have been secreted into the supernatant by your cells carrying the myc-tagged phage protein. This will concentrate the phage, giving you the best chance to detect plaques next time.

1. Harvest 1 ml of cells, if you have that much left, by moving that volume to an eppendorf tube and spinning at full speed in a microfuge for 1 minute (remember to balance your tube). If you do not have a full ml left, then harvest the remaining volume of cells and adjust the volume of PEG in the next step accordingly.
2. Mix 1 ml of supernatant with 1/6th volume (166 ul) 25% PEG 8000 in 2.5M NaCl.
3. Keep on ice for 15 minutes.
4. Spin in a microfuge full speed for 15 minutes.
5. Apirate the supernatant, taking care to leave the pellet of phage undisturbed.
6. Resuspend the phage pellet in 50 ul sterile water.
7. Titer 10 ul of this solution as well as 10 ul of a 10^-2 dilution, consulting your lab notebook or the protocol from day 2 of this module if you need reminding about the procedure.

Part 3: M13.1

This is a rough sketch to refine before we request a DNA synthesis company to compile the program for us. All elements are specified at the Registry of Standard Biological Parts. Proposed refinements can be noted there as well as on the discussion page associated with this day of lab.

genetic element	promoter	RBS	coding
	start synthesis with
gII	BBa_M13102 (need 5' UTR?)	BBa_M13502	BBa_M13002' (modified to remove gene 10 promoter)
gX	BBa_M13110 (need 5' UTR?)	BBa_M13510	BBa_M13010' (modified to remove gene 5 promoter)
gV	BBa_M13105 (need 5' UTR?)	BBa_M13505	BBa_M13005
gVII		BBa_M13507	BBa_M13007' (modified to remove overlap with gene 9 dwnstm)
gIX		BBa_M13509	BBa_M15009' (modified to remove overlap with gene 8 dwnstm)
gVIII	BBa_M13108 (need 5' UTR?)	BBa_M13508	BBa_M13008
	Transcriptional terminator (if M13K07 part, then need to modify to remove gene 3 promoter)
gIII	BBa_M13103	BBa_M13503	BBa_M13003' (modified to remove gene 6 promoter, change GTG start?)
gVI	BBa_M13106	BBa_M13506	BBa_M13006' (modified to remove gene 1 promoter)
gI	BBa_M13101	BBa_M13501	BBa_M13001' (modified to remove gene 11 RBS, gene 4 promoter, RBS, start)
gXI		BBa_M13511	BBa_M13011' (modified to remove gene 4 promoter, RBS, start)
gIV	BBa_M13104 (need 5' UTR?)	BBa_M13504	BBa_M13004'
	M13K07 ori/KanR/p15a ori
	end synthesis

Note: modified parts codon varied to remove direct repeats.

DONE!

For next time

1. Remind yourself what size you expect to see for p8 or p3, depending on which you myc-tagged. Determine which of the kaleidoscope markers you expect to see nearby.
2. Your first draft of your M13 refactoring paper will be due when you arrive in lab next time.

Reagents list

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