Protocol of Western Blotting For High Molecular Weights
Licia Miller Product Manager
High molecular weight western blotting is a modification of the standard western blotting technique specifically designed for the detection and analysis of large molecular weight proteins.
Required Solutions and Reagents
(1) 10× Electrophoresis buffer
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• First dissolve the ingredients in 3.5 L of water and then make up to 5 L.
• When using, dilute to 1× at a ratio of 1:10.
(2) 1× Transfer buffer
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• First dissolve the ingredients in 3.5 L of water and then make up to 10 L.
• Before use, add 10% methanol to the transfer buffer.
(3) Blocking buffer
• 5% NFDM/TBST
(4) 10× TBS
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• Dissolve in deionized water, adjust pH to 7.4 with HCl, and make up to 22.5 L with deionized water.
(5) 1× TBST
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• Add 2.25 L 10× TBS and 22.5 mL Tween-20 and make up to 22.5 L with deionized water.
(6) Separation gel
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(7) Stacking gel
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Stage 1 Sample loading and gel running
Experimental Steps
1. Add equal amounts of protein and molecular weight standards to the wells of an SDS-PAGE gel. Load 20 μg of total protein per lane.
2. Using pre-cooled electrophoresis buffer, run the gel at 150 V for approximately 1.5 hours.
Note: Times and voltages may need to be optimized. Also, unless the antibody datasheet recommends non-reducing conditions, a reducing gel should be used.
Stage 2 Transfer membrane
Experimental Steps
1. Immerse the gel in 1× transfer buffer for 40 minutes.
2. Activate the PVDF membrane with 99.5% methanol for 15 seconds.
3.Immerse PVDF membrane, filter paper, and sponge in 1× transfer buffer for 30 minutes before transfer.
4.Perform wet transfer using pre-cooled transfer buffer at 500 mA for 1 h at 4°C .
5. Wash twice with deionized water for 10 minutes each time and then proceed with antibody staining. Or dry and store temporarily at 4°C.
Phase 3 Antibody staining
Experimental Steps
1. Block the membrane with 5% blocking buffer for 1 hour at room temperature or overnight at 4°C.
2. Wash the membrane with 1× TBST for 10 minutes.
3. Dilute the primary antibody to the recommended concentration in blocking buffer, then pour onto the membrane and incubate at room temperature on a low-temperature shaker for 1 hour.
4. Wash three times with TBST, 10 minutes each time.
5. Immerse the membrane in the conjugated secondary antibody diluted in blocking buffer and incubate at room temperature on a low-temperature shaker for 1 hour.
6. Wash three times with TBST, 10 minutes each time.
7. According to the properties of the conjugated secondary antibody used, select the corresponding signal development methods, such as using darkroom development technology to obtain chemiluminescent images, or using conventional image scanning methods to obtain colorimetric detection images.
For more product details, please visit Aladdin Scientific website.