Application
Western blot
Sample
Human Tissue lysate - whole (Cartilage)
Loading amount
25 µg
Specification
Cartilage
Gel Running Conditions
Reduced Denaturing (4-20% gradient gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Other product details
Dilution
1/1000
Incubation time
2 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: PBSA ,0.05% Tween 20, 5% skimmed milk
Secondary antibody
Name
Non-Abcam antibody was used: Sigma A4914
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/20000
Detection
Detection method
ECL plus
Exposure
30 minute(s) and 0 second(s)
Bands
Specific: Approx 250 and 50 kDa
Additional data
Additional Notes
Anticipated band at approx 50kDa detected. Specificity of antibody allowed relatively long exposure and quite high concentrations of primary and secondary antibodies to be used with negligible background. Good results were also obtained with unreduced samples.
The additional approx 250kDa band is likely to be due to PEDF crosslinked or bound to high molecular weight matrix proteins (collagen, aggrecan) which are present in cartilage lysates. Reduction using beta mercaptoethanol reduced some, but not all high MW staining and increased the intensity of the 50kDa band
Lanes 1-4: Cartilage lysates from 4 osteoarthritic knee replacement donors. Lane C: Positive control (overexpressing cell line)
Samples: 100mg (wet weight) powdered cartilage/ 1ml RIPA buffer, extracted for 24hrs at 4 degrees C. Immobilon membrane.
The additional approx 250kDa band is likely to be due to PEDF crosslinked or bound to high molecular weight matrix proteins (collagen, aggrecan) which are present in cartilage lysates. Reduction using beta mercaptoethanol reduced some, but not all high MW staining and increased the intensity of the 50kDa band
Lanes 1-4: Cartilage lysates from 4 osteoarthritic knee replacement donors. Lane C: Positive control (overexpressing cell line)
Samples: 100mg (wet weight) powdered cartilage/ 1ml RIPA buffer, extracted for 24hrs at 4 degrees C. Immobilon membrane.
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.
DR. John Wardale
Verified customer
投稿 Sep 24 2008