Western blot analysis of extracts from various cell lines using TRAF5 (D3E2R) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human TRAF5 (hTRAF5-Myc/DDK; +) using TRAF5 (D3E2R) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Western blot analysis of extracts from A172 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® TRAF5 siRNA I #60131 (+), or SignalSilence® TRAF5 siRNA II #38805 using TRAF5 (D3E2R) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The TRAF5 (D3E2R) Rabbit mAb confirms silencing of TRAF5 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
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
TRAF5 (D3E2R) Rabbit mAb recognizes endogenous levels of total TRAF5 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His383 of human TRAF5 protein.
TRAFs (TNF receptor-associated factors) are a family of multifunctional adaptor proteins that bind to surface receptors and recruit additional proteins to form multiprotein signaling complexes capable of promoting cellular responses (1-3). Members of the TRAF family share a common carboxy-terminal "TRAF domain", which mediates interactions with associated proteins; many also contain amino-terminal Zinc/RING finger motifs. The first TRAFs identified, TRAF1 and TRAF2, were found by virtue of their interactions with the cytoplasmic domain of TNF-receptor 2 (TNFRII) (4). The six known TRAFs (TRAF1-6) act as adaptor proteins for a wide range of cell surface receptors and participate in the regulation of cell survival, proliferation, differentiation, and stress responses.
TRAF5 regulates signaling through binding to the cytoplasmic domains of TNFR famly members including CD40, CD27, CD30, OX40, and lymphotoxin-β receptor (5-10). Overexpression of TRAF5 induces NF-κB activation. Cytoplasmic aggregates of TRAF5, as well as TRAF2, were reported in Hodgkin-Reed-Sternberg cells, resulting in constitutive NF-κB activation (11).
Studies of TRAF5 deficient mice suggest that it plays an important role in limiting Th2 immune responses that triggers T-cell mediated inflammatory diseases and asthma (12). Further studies indicate that TRAF5 binds to the IL-6 receptor gp130 and negatively controls Th17 differentation (13). In B-cells, TRAF5 negatively regulates toll-like receptor (TLR) mediated cytokine and antibody production (14).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
Tween is a registered trademark of ICI Americas, Inc.