TITLE: Western for Brain Punches
AUTHORS: Olivier George, Lieselot Carrette, Francisco Ramirez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the step-by-step procedure of a western blot; for isolating and probing for proteins from a rat brain punch. </div></div>

[STEPS]
?. [Cell Lysis & Protein Extraction]
Make lysis solutionAdd RIPA an 100X HALT (for end solution of 1X HALT)
?. [Cell Lysis & Protein Extraction]
Add   RIPA/HALT solution to brain punch.
20 µl
?. [Cell Lysis & Protein Extraction]
Homogenize with micropipette (by pipetting up & down and avoiding bubbles).
?. [Cell Lysis & Protein Extraction]
Spin the samples in cold centrifuge at
Centrifuge: 6000 33, 10 min, 4 10
?. [Sample Preperation]
Make a sample buffer 4X Laemmli Sample Buffer +  2-Mercaptoethanol.
45 µl
5 µl
?. [Sample Preperation]
After centrifugeing sample from Part 1, put them
on ice
?. [Sample Preperation]
Take 200 ul  strip tubes and label. Add   of the sample buffer and  of the sample in each tube.
5 µl
15 µl
?. [Sample Preperation]
Repalce strip tube lid.
?. [Sample Preperation]
Incubate these samples in the PCR machine for   at
95 °C
?. [Running Gel]
Take a BioRad gel (with 12 wells if you have 10 smapels) from the fridge. Pull gently to remove the green tape from the bottom of the cassette, and remove the green comb by pulling upward in one smooth motion.
?. [Running Gel]
Insert the gel in the BioRad electrophoresis chamber (with the long side of the gel facing you) and insert the wall on the other side (with the solid side towards the wire).
?. [Running Gel]
Pour the running buffer in the middle and let it overflow until the solution reaches the black line at the bottom.
?. [Running Gel]
Clean wells with a 10 ml syringe and 17 G needle. Denser fluid may be at bottom of wells and needs to be flushed out.
?. [Running Gel]
Take samples out of PCR machine.
?. [Running Gel]
Using gel loading tips, add   ladder to first and last well.
10 µl
?. [Running Gel]
Add   each sample to each well to bottom of wells.
15 µl
?. [Running Gel]
Replace top to electrophoresis chamber (red on left with gel closer to you).
?. [Running Gel]
Run for  at 200 V.
?. [Transfer]
Be sure that transfer buffer and milliQ water for transferring is cold   .
4 °C
?. [Transfer]
Rinse transfer pads with deionized water.
?. [Transfer]
Place transfer pads and filter paper into transfer container and soak in transfer buffer on shaker.
?. [Transfer]
Open the gel cassette using tool
?. [Transfer]
Cut off one corner of the gel with a clean sharp razor blade or scalpel to allow you to orientate the gel.
?. [Transfer]
Cut off top wells, bottom, and sides to remove gel excess.
?. [Transfer]
Assemble blot module.
?. [Transfer]
On anode (+) core place 2 transfer pads, roll the air out with falcon tube and add more transfer buffer on top.
?. [Transfer]
Add 1 filter paper,  roll out air, and more transfer buffer on top
?. [Transfer]
Add 1 membrane
?. [Transfer]
Cut off one corner of the gel mark first well location
?. [Transfer]
Layer gel on top, preventing air from being trapped under
?. [Transfer]
Add 1 filter paper on top of gel, 3-4 transfer pads, roll out air after each one, and add more transfer buffer on top.
?. [Transfer]
Place the ???????cathode (-) core on top of the pads.
?. [Transfer]
Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. Note: blot module fits into the unit in only one way.
?. [Transfer]
Add transfer buffer inside blot module and milliQ water in surrounding area.
?. [Transfer]
Run transfer for   min at 24 V. Do not let sit for an extended period of time after transfer has been completed
?. [Transfer]
During transfer, make 3% BSA solution.
?. [Transfer]
Upon completion of transfer, remove gel (throw away) and put membrane right-side up in a container (e.g., empty micropipette tip container).
?. [Transfer]
Add blot blocker (blocking milk) and leave membrane on shaker for a minimum of
?. [Primary Antibody]
Drain blot blocker from membrane and give a quick rinse with PBST.
?. [Primary Antibody]
Wash membrane in small amount of BSA solution.
?. [Primary Antibody]
Cut top and bottom off membrane around the area of interest, as well as in between the protein of interest and the housekeeping reference protein.
?. [Primary Antibody]
Add each piece of membrane to separate Petri dish.
?. [Primary Antibody]
Pipette  1° antibody into each Petri dish. Incubate  at   .
4 °C
?. [Secondary Antibody]
Pipette out 1° antibody from Petri dishes.(be sure to add the antibodies to their respective tubes for future use)
?. [Secondary Antibody]
Wash in PBST once quickly by hand to remove excess 1° antibody.
?. [Secondary Antibody]
Wash in generous amount of PBST for 5 min 3 times on shaker (some antibodies may need more washing).Wash 1: Wash 2: Wash 3:
?. [Secondary Antibody]
Dilute 2° antibodies with horseradish peroxidase to 1:10,000 (1 ul antibody in 10 ml milk) in falcon tubes or as directed in milk blocking solution.
?. [Secondary Antibody]
Lightly dry membrane strips and place on parafilm (away from workbench edge).
?. [Secondary Antibody]
Pipette diluted 2° antibody to membrane strips.
?. [Secondary Antibody]
Incubate for  at  on a stationary surface.
0 Room temperature
?. [Secondary Antibody]
Wash in PBST once, quickly by hand.
?. [Secondary Antibody]
Wash in PBST for 5 min 4 times on shaker.Wash 1: Wash 2: Wash 3: Wash 4:
?. [Imaging]
Transport blot in Petri dish with PBST for imaging.
?. [Imaging]
Open “Image Lab 5.2.1" and set up imaging parameters.Select Chemi under ApplicationPress Signal Accumulation Setup.Set last image to 900 sec and 15 sec between each image (60 images total).
?. [Imaging]
Drain PBST from Petri dishes.
?. [Imaging]
Add oxidizing reagent and equal amount of enhancing reagent to cover membrane.
?. [Imaging]
Manually shake for   to ensure the whole blot is covered.
?. [Imaging]
After a light dry, put membrane strips into a single piece of binder sheet protector plastic.
?. [Imaging]
Lay inside imager and adjust position using camera to center.
?. [Imaging]
Run program until an good signal occurs for each blot.
?. [Imaging]
Save the photo just prior to oversaturation for each blot.
?. [Imaging]
Turn off imager and computer.
?. [Stripping & Reprobing]
Drain PBST from Petri dishe.
?. [Stripping & Reprobing]
Wash membrane in 1X TBS once, quickly by hand.
?. [Stripping & Reprobing]
Wash membrane in 1X TBS for 5 min 3 times on shaker.Wash 1: Wash 2: Wash 3:
?. [Stripping & Reprobing]
Drain TBS and remove excess liquid from Petri dish with Kimwipe
?. [Stripping & Reprobing]
Immerse blot in Restore Western Blot Stripping Buffer and incubate on shaker for  .
?. [Stripping & Reprobing]
Drain Restore Western Blot Stripping Buffer from Petri dishes.
?. [Stripping & Reprobing]
Wash membrane in 1X TBS for 5 min 5 times on shaker.Wash 1: Wash 2: Wash 3: Wash 4: Wash 5:
?. [Stripping & Reprobing]
Add blot blocker (blocking milk) and leave membrane on shaker for a minimum of
?. [Stripping & Reprobing]
Continue with protocol for primary and secondary antibodies for second protein of choiceFollow protocol through imaging