Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (Saccharomyces cerevisiae)
Loading amount
20 µg
Specification
Saccharomyces cerevisiae
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Other product details
Dilution
1/2500
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 1% BSA/PBST
Secondary antibody
Name
Non-Abcam antibody was used: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate
Host species: Rabbit
Clonality: Monoclonal
Conjugation: Horse Radish Peroxidase
Host species: Rabbit
Clonality: Monoclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/10000
Detection
Detection method
Immun-Star™ WesternC™ Chemiluminescence
Exposure
10 minute(s) and 0 second(s)
Bands
Specific: No specific bands kDa Non-specific: 87, 96, 110 kDa
Positive control
Endogenous Sch9 - Expected size 92Kda
Endogenously HA-tagged Sch9 - Expected size 105KDa
Endogenously HA-tagged Sch9 - Expected size 105KDa
Negative control
sch9 Deletion strain - there should be no band
Additional data
Additional Notes
I tried diluting the Ab 1:500 as recommended by the manufacturer, but I was unable to see any specific bands at the molecular weight expected. Also there was no difference in the non-specific band pattern between the lysate containing the endogenous full-length Sch9 and the lysate with Sch9 deleted.
The Ab did not recognize endogenous Sch9. I tried 6 different set of conditions in an attempt to minimize the high number of nonspecific banding and to optimize the possibility of recognizing the specific band, including varying blocking solutions/primary Ab solutions and dilutions.
The set that I am submitting gave me the lowest level of background banding, but still did not show the specific band.
The Ab did not recognize endogenous Sch9. I tried 6 different set of conditions in an attempt to minimize the high number of nonspecific banding and to optimize the possibility of recognizing the specific band, including varying blocking solutions/primary Ab solutions and dilutions.
The set that I am submitting gave me the lowest level of background banding, but still did not show the specific band.
Abcam response
Thank you for this valuable feedback. This antibody has only ever been used to detect recombinant fragments of SCH9 and may not be suitable for detecting SCH9 in Saccharomyces cerevisiae .
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.
Abcam user community
Verified customer
提交于 Feb 28 2013