Western blot analysis of extracts from various cell lines and tissues using Complement C4-A/C4-B (E6J7W) Rabbit mAb (upper) and β-Tubulin (D2N5G) Rabbit mAb #15115 (lower).
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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
Complement C4-A/C4-B (E6J7W) Rabbit mAb recognizes endogenous levels of total C4-A/C4-B protein. The predicted epitope of Complement C4-A/C4-B (E6J7W) Rabbit mAb is the N-terminus of the β-subunit of the C4-A/C4-B protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro148 of human C4-A/C4-B protein.
Complement is a collection of soluble proteins secreted in the blood and other body fluids (1). As part of the innate immune system, complement proteins are involved in the clearance of pathogens and damaged cells in a process called opsonization, which results in the coating of a pathogen with antibodies and/or complement proteins to facilitate phagocytosis of debris or foreign pathogens. Complements are activated by a cascade of cleavage reactions, triggered initially by pattern recognition receptor-mediated detection of pathogens/debris. The cascade of cleavage-mediated products activate three distinct effector pathways, including inflammation, phagocytosis, and membrane attack that represent a coordinated defense of the host organism. Several complement proteins are encoded in the mammalian genome, designated by the capital letter C followed by a number, in order by their discovery. Complement C4-A and C4-B, encoded by the C4A and C4B genes, respectively, are the non-enzymatic components of C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. The C4A/C4B genes generate C4-A/C4-B polypeptide proteins, composed of an alpha, beta, and gamma-chain subunits linked by disulfide bonds. The polypeptide protein can be further cleaved to generate a cascade of fragments to initiate the innate immune response. In addition to the innate immune response, several components of the complement system, including C4-A, have been implicated in brain development, as well as neurodevelopmental and neurodegenerative diseases. The complement system plays a role in microglial-dependent synapse pruning of excess synapses during development, particularly in the visual system (2). Additionally, complement-mediated synaptic pruning may also contribute to psychiatric diseases such as schizophrenia (3,4).
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