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Recent Antibody Validation Data

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Organization/institution Johns Hopkins University
Validation data ID 112
Submission date 2017-02-10
Product name Rabbit polyclonal SEPT2 antibody (A5801)
Product type Primary antibody
Product catalog No. A5801
Supplier ABclonal
Application type Western Blot
Antibody specificity rating Good
Antibody overall rating Excellent
Your comments

We tested the septin (SEPT) antibody on western blots with total lysates of human retinal epithelial cells (hTert-RPE1, ATCC). The antibody showed strong clean band at the expected molecular weight (41kDa), indicating that it is likely specific. We did not test the specificity of the antibody by CRISPR, RNAi or otherwise, which is the only reason for only the "good" rating of specificity.

Materials Total lysates of human retinal epithelial cells (hTert-RPE1, ATCC)
Treatment of materials Cells were trypsinized, washed in DPBS and lysed by boiling in 2x SDS PAGE sample buffer without b- mercaptoethanol and bromphenol blue. Total protein conc was determine by Micro BCA. Samples were mixed with 1x full SDS PAGE sample buffer (with the dye and b- mercaptoethanol) to concentration of 2mg/ml and stored at -80C.
Gel electrophoresis BioRad minigels 4-20%, 15 wells, with 5-20 micograms/well
Transfer BioRad Transblot Turbo, PVDF membrane, 7 min transfer
Blocking 5% nonfat dried milk or BioRad blocker in TTBS, 30 min room temperature
Membrane wash 0.05% Tween 20 in TBS, pH7.4 (TTBS), 3 times between the first and second antibody, at least 5 times before ECL detection
Primary antibody incubation 1:1000, overnight at 4C
Secondary antibody
Name Supplier/brand Incubation
anti-Rabbit IgG, HPX -linked Cell Signaling Technology 20-30 min at room temp
Loading control antibody (optional)
Name Supplier/brand
mouse anti- b-actin 1:10 000 GenScript
Sample Loading Information
Lane No. Sample Loading amount Primary Antibody dilution Secondary Antibody dilution Target band (KD) Exposure Time
1 hTERT-RPE1 cells 5 ug 1:1000 1:5000 41 10s
2 hTERT-RPE1 cells 10 ug 1:1000 1:5000 41 10s
3 hTERT-RPE1 cells 20 ug 1:1000 1:5000 41 10s
Result figure Result figure
Figure description

Immunoblotting detection of septin (SEPT) in total lysates of hTERT-RPE1 cells

Product information
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