Western blot analysis of extracts from various cell lines using IgM Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, IgM heavy chain protein expression is not detected in either MOLT-4 or HPB-ALL cells.
Western blot analysis of extracts from Daudi cells, untreated (-) or treated with PNGase F (+), using IgM Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. 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
IgM Antibody recognizes endogenous levels of total IgM heavy chain protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of the human IgM heavy chain constant region. Antibodies are purified by protein A and peptide affinity chromatography.
Immunoglobulin M (IgM) is one of the five major human immunoglobulin isotypes and is the first antibody to be secreted by plasmablasts in a humoral immune response after exposure to antigen. Structurally, IgM is the largest immunoglobulin and predominantly exists in pentameric form when secreted (1). Alternative splicing of the IgM heavy chain mRNA can generate an alternative form of the antibody, which facilitates its insertion into the plasma membrane of B-cells to function in antigen recognition. IgM is the first Ig isotype to appear on the surface of developing B-cells and is a major component of the B-cell antigen receptor (BCR) signaling complex, which drives B-cell survival and proliferation upon antigen-induced ligation (2,3). Research studies have shown that defects in the assembly of the BCR account for the low levels of surface IgM expression in B-chronic lymphocytic leukemia (4,5).
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