Application
Western blot
Sample
Human Cell lysate - whole cell (Monocytes and Macrophages)
Loading amount
100000 cells
Specification
Monocytes and Macrophages
Treatment
Untreated monocytes or monocytes treated for 5 days with PMA plus 1 day PMA and hydrocortisone to differentiate them to macrophages
Gel Running Conditions
Reduced Denaturing (Pierce Precise 4 - 20 % gradient protein gels)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Other product details
Dilution
1/250
Incubation time
9 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5 % milk blocking buffer
Secondary antibody
Name
Non-Abcam antibody was used: Santa Cruz bovine anti-mouse IgG
Host species: Cow
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Cow
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/1000
Detection
Detection method
ECL+
Exposure
5 minute(s) and 0 second(s)
Bands
Specific: 200 kDa Non-specific: 180 kDa
Positive control
Differentiated macrophages (right hand lane)
Negative control
Peripheral blood monocytes selected by adherence to plastic (left hand lane)
Additional data
Additional Notes
Lower band appears to be the unglycosylated form of this very large protein (we think), whilst the upper band appears to be the more abundant / specific glycosylated form. This form also appears to be expressed at low levels in adherent monocytes as well.
NB. This is a very large protein and requires addition care to ensure that it transfer, thus we used gradient gels.
NB. This is a very large protein and requires addition care to ensure that it transfer, thus we used gradient gels.
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 Sep 23 2009