TITLE: Western blotting of hypothalamic proteins
AUTHORS: Kira V. Derkach, Irina O. Zakharova, Inna I. Zorina, Andrey A. Bakhtyukov, Irina V. Romanova, Liubov V. Bayunova, Alexander Shpakov

[STEPS]
?. [Sample preparation:]
Homogenize a piece of hypothalamus tissue (½ of hypothalamus) in the 1X Tissue Lysis Buffer in the ratio of 1:10.
?. [Sample preparation:]
Keep homogenate on ice for 30 min.
?. [Sample preparation:]
Centrifuge the homogenate at 14 000g for 10 minutes (4 °C).
?. [Sample preparation:]
Transfer the supernatant to a new tube and discard the pellet.
?. [Sample preparation:]
Remove 3x3 μl of supernatant to measure the protein concentration using modified method of Lowry. Mix an aliquot of lysate containing 300 μg of protein with the Lysis Buffer to adjust to a volume of 50 μl, and then add 50 μl of 2X SDS Sample Loading Buffer.
?. [Sample preparation:]
Warm the samples for 5 min at 95 °C.
?. [Sample preparation:]
Cool at the room temperature for 5 min.
?. [Sample preparation:]
Centrifuge the samples for 5 minutes using the Microfuge 22R centrifuge (Beckman Coulter, USA).
?. [Sample preparation:]
Load up to 10 μl of sample to each well of a 1.5 mm thick gel. Guidelines for choosing the gel percentages are based on the protein size to be detected: 8.5% gel for the analysis of 80-200 kDa proteins, and 9-14% gel for the analysis of 20-65 kDa proteins.
?. [Sample preparation:]
Set gel running conditions at 75 V at the beginning of electrophoresis procedure, gradually increasing it to 95 V, using SDS Running Buffer.
?. [Sample preparation:]
Transfer the proteins to a nitrocellulose membrane at 100 V for 1 hour, using Transfer Buffer.
?. [Membrane blocking:]
Disassemble the transfer sandwich, remove the blotted membrane, wash the membrane 3-4 times with distilled water and immediately place in the Blocking Buffer consisting of 5% non-fat dry milk/TBST.
?. [Membrane blocking:]
Incubate the blot for 30 min-1 hour at the room temperature with the agitation.
?. [Antibody Incubation:]
The primary antibody is diluted according to the manufacturer’s recommendations. In the most cases, the antibodies are diluted in the 5% BSA/TBS-T in the ratio of 1:1000.
?. [Antibody Incubation:]
Place the membrane in the primary antibody solution and incubate overnight at 4°C with the agitation.
?. [Antibody Incubation:]
Wash for 5 min with the TBS containing 0.1% Tween-20 (wash 1/3).
?. [Antibody Incubation:]
Wash for 5 min with the TBS containing 0.1% Tween-20 (wash 2/3).
?. [Antibody Incubation:]
Wash for 5 min with the TBS containing 0.1% Tween-20 (wash 3/3).
?. [Antibody Incubation:]
Incubate the membrane for 1 hour at the room temperature with horseradish peroxidase (HRP)‐conjugated secondary antibodies diluted to 1:1000–1:5000 in 5% non-fat dry milk/TBS‐T.
?. [Antibody Incubation:]
Wash for 5 min with the TBS containing 0.1% Tween‐20 (1/3).
?. [Antibody Incubation:]
Wash for 5 min with the TBS containing 0.1% Tween‐20 (2/3).
?. [Antibody Incubation:]
Wash for 5 min with the TBS containing 0.1% Tween‐20 (3/3).
?. [Antibody Incubation:]
Wash for 5 min with the TBS without Tween‐20 (1/2).
?. [Antibody Incubation:]
Wash for 5 min with the TBS without Tween‐20 (1/2).
?. [Antibody Detection:]
Incubate the membrane (protein side up) with 10 ml of ECL (enhanced chemiluminescence substrate) for 1 min. The final volume required is 0.125 mL/cm2.
?. [Antibody Detection:]
Drain off the excess of detection reagent, wrap up the blots, and gently smooth out any air bubbles.
?. [Antibody Detection:]
Place the wrapped blots, protein side up, in an X-ray film cassette and expose to X-ray film. Exposures can vary from 5 seconds to 60 minutes.