Western blot analysis of recombinant Human Granulocyte Macrophage Colony Stimulating Factor (hGM-CSF) #8922 using GM-CSF (E8I1V) Rabbit mAb.
Western blot analysis of extracts from NK-92 cells, untreated (-) or treated (+) with TPA #4174 (80 nM, 5 hr), Ionomycin #9995 (3 μM, 5 hr), and Brefeldin A #9972 (300 ng/mL, last 4 hr of stimulation), using GM-CSF (E8I1V) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
|MW (kDa)||15, 18, 22|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
GM-CSF (E8I1V) Rabbit mAb recognizes endogenous levels of total GM-CSF protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro29 of human GM-CSF protein.
GM-CSF is produced by activated T cells, NK cells and macrophages (1,5). Target cells include granulocyte, monocyte precursors and subsets of differentiated myeloid cells (1,2,3). Many target cells require GM-CSF for survival. GM-CSF induces proliferation, is involved in hematopoietic differentiation of dendritic cells, and is a key factor in differentiation pathways leading from stem cells. GM-CSF activates effector functions of myeloid cells, thereby linking adaptive and innate immunity and in turn may boost anti-tumor immunity (4). GM-CSF receptor is composed of GM-CSFRα and the common β chain, βC, which is also utilized by IL-3 and IL-5 (1). Binding of GM-CSF initiates the Jak2, Stat5, and PI3K/Akt pathways (1).
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