BRCA2 (phospho T3349) antibody (ab36459)

All antibodies work in different combinations of applications, species and samples, so we encourage our customers to write a short Abreview, so that future customers can make an informed decision about the usefulness of a particular antibody for their specific experiments.

Submit an Abreview and earn Abpoints

Anonymous (click to contact this user) Abreview submitted on 17 September 2008
Application:	Western blot
BRCA2 (phospho T3349) antibody (ab36459)
Sample	Human Cell lysate - whole cell (MDA-MB-468 cell line)
Loading amount:	100 µg
Specification	MDA-MB-468 cell line
Gel Running Conditions:	Reduced Denaturing (6% gel)
Blocking step:	Milk as blocking agent for  1 hour  · Concentration: 5 %
Primary Antibody
Dilution	1/100
Incubation time:	12 hours  Temperature: 4°C
Secondary Antibody
Name:	Non-Abcam Antibody was used: anti-rabbit antibody Conjugation: HRP
Dilution:	1/1000
Detection
Detection method:	ECL
Exposure:	30 seconds
Bands:	Specific: 460 kDa
Additional Data
Rating	Good
Additional notes	1. Sample preparation: MDA-MB-468 cells were lysed by using CelLytic MT reagent (sigma C3228) containing Protease Inhibitor Cocktail (sigma P8340) and Phosphatase Inhibitor Cocktail (sigma P2850). The protein concentration was determined by protein assay ( Bio-Rad 500-0121). Then cell lysate was mixed together with same volume of Laemmli sample buffer (Bio-Rad 161-0737) with ß-captoethenal. Sample was boiled for 5 minutes. 2. Electrophoresis: 6% gel was prepared. 100 microgram sample was loaded to gel. The running voltage was 80 volt for 30 minutes, 120 volt for about 2 hours. 3. Transfer: Gel was soaked in the prechilled transfer buffer (20% methanol in running buffer) for 15 minutes before transferring. Nitrocellulose membrane (GE 1212632) was used for transfer. Then the sandwich was made. The transfer voltage was 80 volt for 2 hours. Keep the whole system in cold room during transferring. 4. Probing: Membrane was blocked in 5% milk in TBST for 1 hour at room temperature. Primary antibody was then added to blocking buffer (1:100 dilution), incubation overnight at 4 degree. The membrane was washed by TBST for 4 times. Then pour secondary antibody ( Santa cruz sc-2004) solution to the membrane (1:1000 dilution) and incubate for 1 hour at room temperature. Wash by TBST for 4 times. 5. Detection: Amersham ECL™ Western Blotting Detection Reagents (RPN2209) was used. Film was exposed to the membrane for 30 seconds. Then develop the film. Conclusion: This antibody can detect BRCA2 T3349 in MDA-MB-468 cell line.
Image	BRCA2 (phospho T3349) antibody for WB (Human) Copyright image used under license