Application
Western blot
Review text: The blot image provided in this review has mouse Dorsal Striatum loaded at 70ug in lanes 2-7 and the P35/P25-GST lysate in .5, 1 and 3 ug in lanes 8-10. The .5ug amount appears to be the least necessary to produce both the p35-GST and p25-GST bands. The p25-GST band is much stronger than the p35-GST band. A goat polyclonal anti-GST antibody was used from GE Health sciences. Therefore, lanes 8-10 have a yellow band because of co-labeling of p35/p25 and the GST tag. There was some nonspecific binding with the p35/p25-GST lysate due to such a high amount being used. These bands are found mainly below the p25 band. There was also nonspecific binding with the anti-GST antibody within the p35/p25-GST lysate but not the Dorsal Striatum lysates. Only p35 appears in the Dorsal Striatum lanes because the 10% gel was not able to resolve p25 because it is too light. 12.5% gels have been used to resolve both p35 and p25 in previous studies, but this percent gel would not have been able to resolve the GST tagged proteins. A gradient gel would be necessary.
Sample: Mouse Cell lysate - whole cell (Brain)
Loading amount: 70 µg
Specification: Brain
Gel Running Conditions: Reduced Denaturing (10% gel)
Blocking step: Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Other product details
Incubation time: 18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% Milk
Primary antibody (in addition to 'p35 + p25 protein (Tagged)')
Name: Non-Abcam antibody was used: CST anti-p35/p25
Host species: Rabbit
Clonality: Monyclonal
Conjugation: Other
Dilution: 1/1000
Secondary antibody
Name: Non-Abcam antibody was used: Licor 700
Host species: Donkey
Clonality: Polyclonal
Conjugation: IRDye® 700DX
Detection
Detection method: Licor
Exposure: 1 hour(s), 0 minute(s) and 0 second(s)
Bands: Specific: 59 and 49 kDa Non-specific: several below 40 kDa
Positive control: The p35/p25-GST lysate served as a positive control
Negative control: None
Sample: Mouse Cell lysate - whole cell (Brain)
Loading amount: 70 µg
Specification: Brain
Gel Running Conditions: Reduced Denaturing (10% gel)
Blocking step: Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Other product details
Incubation time: 18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% Milk
Primary antibody (in addition to 'p35 + p25 protein (Tagged)')
Name: Non-Abcam antibody was used: CST anti-p35/p25
Host species: Rabbit
Clonality: Monyclonal
Conjugation: Other
Dilution: 1/1000
Secondary antibody
Name: Non-Abcam antibody was used: Licor 700
Host species: Donkey
Clonality: Polyclonal
Conjugation: IRDye® 700DX
Detection
Detection method: Licor
Exposure: 1 hour(s), 0 minute(s) and 0 second(s)
Bands: Specific: 59 and 49 kDa Non-specific: several below 40 kDa
Positive control: The p35/p25-GST lysate served as a positive control
Negative control: None
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.
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提交于 Apr 08 2011