M465:Western blots part I

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A visual representation of loading protein sample onto a polyacrylamide gel
Coommassie stained protein gel showing a visual representation of protein separation by weight
Traditional ECL image utilizing a chemiluminescent substrate to react with HRP, producing light that exposes the film in a band pattern

A protein blot, also known as the immunoblot or western blot, is a method of semiquantitative determination of protein expression. Crude lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current passing through the gel will then propel the proteins through the gel, with the largest proteins traveling the slowest. This results in the proteins being roughly separated by their mass. The proteins are then transferred from the gel onto a membrane (often nitrocellulose or PVDF), which is then incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can then specifically detect the protein of interest.

Detecting Wolbachia using western blot

We will ask if the insects we collected at the winery are infected with Wolbachia by probing an immunoblot with antibody specific to Wolbachia. We will use the antibody anti-wsp. Before we can get to that point, however, we must run our protein lysates out on a polyacrylamide gel and then transfer the proteins to a membrane. This is part I of the western blot, which we will complete today.

use gloves throughout!

Protein Gel Electrophoresis

1. Take 1 insect in a 1.5 mL tube and add 200 ul of lysis buffer.
2. Grind the insect in lysis buffer (20 strokes with a pestle, remember to NOT discard used pestles).
3. Gently spin down the debris with a tabletop centrifuge (1 minute spin at 600 g).
4. Remove 20 ul of the sample and place in a new, 1.5 mL tube.
5. To this 20 ul sample, add 10ul of 2x Laemmlil loading buffer. Cap the tube with a special colored top to make sure that they do not pop open (provided) and place the tube at 95C for 5 minutes.
6. Gently spin down the tube to get rid of the condensation on the lid
7. Assemble your gel as directed by your instructor.
8. Using gel loading tips, load 25 ul of your sample into the second well (leave the first well empty for the ladder). We will use one gel per pair of students so make sure that you keep notes as to which sample is where!
9. Run the gel until the loading dye is in the bottom 1/4 of the gel (120 V for 1 hour).

Protein Transfer to Membrane

1. Make up the tris-glycine-methanol transfer buffer (you will need 1.5L of this for each pair of gels so make up one per bench).
2. In a 2 L graduated cylinder, measure out the Tris-Glycine buffer (1.250L). Pour this buffer into a 2L flask.
3. Using the same 2L graduated cylinder, measure out 250 mL of methanol. Add this to the buffer in the 2L flask.
4. When your gels are done running, you will create a “blot sammich” by following the protocol below.
5. Grab two tupperware containers per bench (one pair per four students). To the first, add 100 mL of methanol. To the second, add 100 mL of transfer buffer.
6. Assemble 2 fiber pads, a roller, the apparatus for transfer, two membranes, the transfer bucket and 4 pieces of whatman filter paper.
7. Put the pads and whatman papers into the second tupperware containing transfer buffer. Put the membranes into the first tupperware container containing methanol alone (submerge!).
8. Assemble your “blot sammich” as shown below and fill your transfer buckets with the transfer buffer.

Gel Sandwich Depiction

9. We will run for this transfer overnight on ice at 40 V. Your instructor will come in and put your membranes in the appropriate primary antibody so that we can continue with protocol.

Clean up:

1. Wipe down all surfaces.
2. Dump all excess buffers.
3. Return all used pestles and empty containers to the front of the room.