“On Western blots with lysates of human retinal epithelial cells, the antibody weakly detects a band at approximately the expected size (18kDa) and several weaker bands and higher molecular weights. Although we tried this antibody 3 times with the same samples and conditions, it "worked" in this manner only the first time. No signal at all was detected on blots from the second and third repeat, although the antibody concentration was increased from 1:1000 to 1:200. Possibly, this particular batch is not stable.” Figure: Immunoblotting detection Antibody Customer Review: STJ92371 Anti-Cofilin Antibody Antibody Specificity: Antibody Rating: Protocol 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 concentration 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. E.g. 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 probing: 1:1000 dilution at 4 degree Celsius overnight. 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. Secondary antibody probing : 20-30 minutes at room temperature (1:5000 dilution). 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. Visualization: ECL visualization for 300 seconds. Cell Line: Total lysates of human retinal epithelial cells (hTert-RPE1, ATCC) Method of validation: Western Blot Primary Antibody: STJ92371 Anti-Cofilin Antibody Secondary Antibody anti-Rabbit IgG, HPX - linked Reference Antibody Beta Actin Dilution ratio: 1:1000 Gel electrophoresis information: BioRad minigels 4-20%, 15 wells, with 5-20 micograms/well Transfer information: BioRad Transblot Turbo, PVDF membrane, 7 min transfer Lane No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualiz ation time 1 wt hTert RPE1 cells 5ug STJ92371 Anti- Cofilin Antibody 1:1000 1:5000 ~18kDa 300 seconds 5 wt hTert RPE1 cells 10ug 9 wt hTert RPE1 cells 20ug

Western Blot Antibody Customer Review for Anti-Cofilin Antibody (STJ92371)

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Page 1: Western Blot Antibody Customer Review for Anti-Cofilin Antibody (STJ92371)

Result“On Western blots with lysates of human retinal epithelial cells, the antibody weakly detects a band at approximately the expected size (18kDa) and several weaker bands and higher molecular weights. Although we tried this antibody 3 times with the same samples and conditions, it "worked" in this manner only the first time. No signal at all was detected on blots from the second and third repeat, although the antibody concentration was increased from 1:1000 to 1:200. Possibly, this particular batch is not stable.” Figure: Immunoblotting detection of cofilin in total lysates of hTERT-RPE1 cells. – External Laboratory

Antibody Customer Review: STJ92371 Anti-Cofilin Antibody

Antibody Specificity: Antibody Rating:

ProtocolTreatment 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 concentration 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.

E.g. 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 probing: 1:1000 dilution at 4 degree Celsius overnight.

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.

Secondary antibody probing: 20-30 minutes at room temperature (1:5000 dilution).

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.

Visualization: ECL visualization for 300 seconds.

Cell Line:Total lysates of human retinal epithelial cells (hTert-RPE1,

ATCC)Method of validation: Western Blot

Primary Antibody: STJ92371 Anti-Cofilin Antibody