This experiment describes the preparation of SDS-PAGE protein samples.
Operation method
SDS-PAGE protein sample preparation
Materials and Instruments
SDS-PAGE Loading Buffer ddH2O 30% Acrylamide Mix 1.5M Tris(pH8.8) 10% SDS 10% Ammonium Persulfate TEMED Tris-Glycine Electrophoresis Buffer Bromophenol Blue Kaomas Brilliant Blue R-250 Staining Solution Kaomas Brilliant Blue Decolorizing Solution Move experimental procedure For more product details, please visit Aladdin Scientific website.
Centrifuges Cold chambers Water baths Electrophoresis units Micropipettes Horizontal shakers Filter paper
The collected samples were added to an equal volume of 2×SDS-PAGE Loading Buffer, boiled for 5 min, ice bath for 2 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was stored at -20℃.
SDS-PAGE protein electrophoresis (refer to Molecular Cloning Guide) (Sambrook, 2002).
1) Load clean and dry glass plates according to the instructions of the electrophoresis device.
2) Preparation of separation gel
Prepare 10 mL of 12% separation gel according to the following composition:
ddH2O 3.3 mL
30% acrylamide mixture 4.0 mL
1.5 M Tris (pH 8.8) 2.5 mL
10% SDS 0.1 mL
10% ammonium persulfate 0.1 mL
TEMED 0.004 mL
Total volume 10 mL
The components were quickly vortexed and mixed after addition, and carefully injected into the prepared glass plate gaps with a micropipette, leaving enough space for the cumulus gel. Gently add a thin layer of water to the top layer to seal the top to prevent the inhibitory effect of oxygen in the air on the gel polymerization. When gel polymerization was complete, the covering layer of water was poured off, the top of the gel was washed with water several times, and the top of the gel was blotted dry with filter paper.
3) Preparation of cumulus gel
Prepare 2 mL of 5% cumulus gel according to the following composition:
ddH2O 1.4 mL
30% acrylamide mixture 0.33 mL
1.0 M Tris (pH 6.8) 0.25 mL
10% SDS 0.02 mL
10% ammonium persulfate 0.02 mL
TEMED 0.002 mL
Total volume 2 mL
After each component is added, vortex it quickly and mix it well. Use a micropipette to fill it onto the separator gel, and when it is full, insert the spiking comb carefully, avoiding air bubbles as much as possible.
(4) After the cumulus gel has solidified, carefully remove the comb.
5) Fix the gel on the electrophoresis device, add a sufficient amount of 1 × Tris-glycine electrophoresis buffer, and add 20 μL of each sample into the spiking well.
6）When the samples are electrophoresed in the cumulus gel, use a voltage of 80V. After the bromophenol blue band enters the separation gel, increase the voltage to 120V and continue electrophoresis until the bromophenol blue band reaches the bottom of the separation gel and starts to swim out of the bottom surface of the gel, then turn off the power.
(7) Remove the gel, soak it in at least 5 times the volume of Caulophyllum blue R-250 staining solution, and place it on a horizontal shaking table at room temperature for at least 4 h. After that, take out the stained gel and recycle the staining solution for reuse, soak the gel in Caulophyllum blue decolorizing solution and decolorize it for 4-8 h on a horizontal shaking table, and change the decolorizing solution for 3-4 times during the time until the gel decolors the bands until they are clear, and then observe the results and record them. The results were observed, recorded and photographed.