Application
Western blot
Sample
Schizosaccharomyces pombe Recombinant protein (N/A)
Loading amount
10 µg
Specification
N/A
Gel Running Conditions
Reduced Denaturing (15%)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C
Other product details
Dilution
1/250
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 20°C · Diluent: 3% Milk + PBST
Secondary antibody
Name
Non-Abcam antibody was used: Anti-Mouse IgG –Alkaline Phosphatase
Host species: Goat
Clonality: Polyclonal
Conjugation: Alkaline Phosphatase
Host species: Goat
Clonality: Polyclonal
Conjugation: Alkaline Phosphatase
Dilution
1/10000
Detection
Detection method
BCIP
Exposure
4 hour(s), 0 minute(s) and 0 second(s)
Bands
Specific: 15 kDa Non-specific: Multiple kDa
Positive control
Coomassie Gel ran with purified recombinant Cam1 and Cam2 from S.pombe to ensure was detecting the specific purified bands. Control using human CaM which has been reported detect.
Negative control
N/A
Additional data
Additional Notes
Coomassie stain shows proteins are present. Proteins transferred onto PVDF with Methanol using a semi dry blotting system. Membrane was Ponceau stained to ensure proteins had transferred (not shown). All bands corresponded to the Coomassie bands.
There is a very faint band in the hCam and the Cam2 samples, however these developed after 4 hours and are much less intense than seen on the coomassie gel.
Varying conditions and concentrations were used, (1/5000, 1/2500, 1/1000, 1/500 and 1/250) and blocking with milk/BSA and also using TBST or PBST. The above gave the only results.
Even the human CaM band is not very clear. This cannot be used as a way to detect any calmodulin in fission yeast.
There is a very faint band in the hCam and the Cam2 samples, however these developed after 4 hours and are much less intense than seen on the coomassie gel.
Varying conditions and concentrations were used, (1/5000, 1/2500, 1/1000, 1/500 and 1/250) and blocking with milk/BSA and also using TBST or PBST. The above gave the only results.
Even the human CaM band is not very clear. This cannot be used as a way to detect any calmodulin in fission yeast.
Abcam response
Thank you for your valuable review, the first we have for detection of fission yeast calmodulin with this antibody. We expect a stronger signal at 17 kDa for calmodulin, at least for the human recombinant sample. The monoclonal was generated against calmodulin from the slime mold D. discoideum and reactivity with human calmodulin was added to the datasheet based on the 2007 reference, Boraldi F et al. PubMed: 17904921.
The reviewer has confirmed that a Coomassie stain of the S. pombe recombinant Cam2 preparation in an SDS-PAGE gel shows a primary band at 17 kDa that is more intense than other bands, indicating a higher purity than the blot suggests.
The reviewer has confirmed that a Coomassie stain of the S. pombe recombinant Cam2 preparation in an SDS-PAGE gel shows a primary band at 17 kDa that is more intense than other bands, indicating a higher purity than the blot suggests.
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 Mar 09 2011