Application
Western blot
Sample
Human Purified protein (human plasma)
Loading amount
50 µg
Specification
human plasma
Treatment
Plama was separated into different bands by agarose electrophoresis, and alphaHDL was cut and protein was extracted for 2nd dimensional SDS-PAGE
Gel Running Conditions
Non-reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Other product details
Dilution
1/10000
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 22°C · Diluent: blocking buffer
Secondary antibody
Name
Abcam antibody:Donkey anti-Goat IgG H&L (AP) secondary antibody
Dilution
1/2500
Detection
Detection method
AP
Exposure
5 minute(s) and 0 second(s)
Bands
Specific: 31 kDa Non-specific: 80, 100, 200 kDa
Positive control
Pure human apoA-I from Sigma
Negative control
Pure human apoA-II from Sigma
Additional data
Additional Notes
Because of big amount of albumin ( overload), it also shows faint band.
1. Plasma was separated in 1st dimensional agarose elephoresis,
2.AlphaHDL band was cut from agarose gel, proteins were extracted from it, then were loaded on 2nd dimensional SDS-PAGE (The gel is 1D gel, but it is second dimensional separation for the proteins. Since in the first dimensional separation, proteins and particles were separated by charge and in 2nd dimensional separation they were separated by size).
3. Proteins were trasfered from gel to membrane by pressure distribution
4. After transfer, SDS-PAGE was stained with silver kit to compare with western bloting result which are stained with AP stain kit.
5. Molecular marker was Kaleidoscope prestained standards from Bio-Rad.
1. Plasma was separated in 1st dimensional agarose elephoresis,
2.AlphaHDL band was cut from agarose gel, proteins were extracted from it, then were loaded on 2nd dimensional SDS-PAGE (The gel is 1D gel, but it is second dimensional separation for the proteins. Since in the first dimensional separation, proteins and particles were separated by charge and in 2nd dimensional separation they were separated by size).
3. Proteins were trasfered from gel to membrane by pressure distribution
4. After transfer, SDS-PAGE was stained with silver kit to compare with western bloting result which are stained with AP stain kit.
5. Molecular marker was Kaleidoscope prestained standards from Bio-Rad.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Dr. xuefei li
Verified customer
投稿 Sep 19 2007