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ReviewID: xxx AbID: 97532, Anti-Human intestinal alkaline phosphatase antibody Rating: Inconclusive Image: Anti-Human intestinal alkaline phosphatase antibody for Western blot (Rat) Sample: Species: Rat Type: Tissue lysate - whole Loading amount: 25 µg Specification: small intestine Application: Application: Gel Running Conditions: Reduced Denaturing (8 % gel) Blocking step: 3% BSA, 5% Milk, Blocking Buffer for fluorescent Western Blotting as blocking agent for 1 hour Blocking Concentration: 3% Other detail: Dilution: 1/500 Incubation time: 16 hours at 4°C Diluent: BSA, Milk or Blocking Buffer Secondary Antibody: Name: Non-Abcam Antibody was used: IRDye 800CW conjugated goat (polyclonal) anti-rabb Conjugation: IRDye® 800CW Dilution: 1/5000 Detection: Detection method: Odyssey Western blot detection, Immunflu Exposure: 1 minute and 0 seconds Bands: Specific: 0 kDa Non-specific: 48, 70 kDa Additional Data: Additional Notes: Type of membran used: PVDF for fluorescence Legend: A: Blocking and 1. + 2. antibody dilution: 3 % BSA B: Blocking and 1. + 2. antibody dilution: Blocking Buffer for fluorescent Western Blotting (Rockland) C: Blocking and 1. + 2. antibody dilution: 5 % Milk M: molecular weight markers 1 = rat small intestine jejunum (proximal) 2 = rat small intestine jejunum (distal) 3 = rat small intestine ileum
Asked on May 14 2012
Thank you for submitting an Abreview of ab97532. As you may have noticed, your review has now been published on our website.
Since you obtained poor results using the antibody in an untested species (rat), we would like to follow up on this.
Based on the information in your review, I feel that altering the following step in your protocol may help improve your results:
- try using a hman sample as positive control
- check the secondary if it could contribute to non-specific bands
- load more protein (e.g. 50 ug)
- use more primary antibody (1/200)
- the mature rat protein might run between 28-35 kDa (see UniProt database: http://www.uniprot.org/uniprot/P15693)
If you decide to follow these suggestions, please let me know if they are helpful.
I look forward to your reply.
Answered on May 14 2012