Western blot analysis of extracts from MCF 10A cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Toll-like Receptor 3 siRNA I #6236 (+), using Toll-like Receptor 3 (D10F10) Rabbit mAb #6961 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Toll-like Receptor 3 (D10F10) Rabbit mAb confirms silencing of Toll-like Receptor 3 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with human TLR3 (+), using Toll-like Receptor 3 (D10F10) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from HT-29 cells, untreated or following transfection with pIpC (100 μg/ml; overnight), using Toll-like Receptor 3 (D10F10) Rabbit mAb.Learn more about how we get our images.
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
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.
Toll-like Receptor 3 (D10F10) Rabbit mAb recognizes endogenous levels of total TLR3 protein. A band is detected at 75 kDa in some cell lines/tissues which is of unknown origin.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val495 of human TLR3 protein.
Members of the Toll-like receptor (TLR) family, named for the closely related Toll receptor in Drosophila, play a pivotal role in innate immune responses (1-4). TLRs recognize conserved motifs found in various pathogens and mediate defense responses (5-7). Triggering of the TLR pathway leads to the activation of NF-κB and subsequent regulation of immune and inflammatory genes (4). The TLRs and members of the IL-1 receptor family share a conserved stretch of approximately 200 amino acids known as the Toll/Interleukin-1 receptor (TIR) domain (1). Upon activation, TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains, including myeloid differentiation factor 88 (MyD88), MyD88-adaptor-like/TIR-associated protein (MAL/TIRAP), Toll-receptor-associated activator of interferon (TRIF), and Toll-receptor-associated molecule (TRAM) (8-10). This association leads to the recruitment and activation of IRAK1 and IRAK4, which form a complex with TRAF6 to activate TAK1 and IKK (8,11-14). Activation of IKK leads to the degradation of IκB, which normally maintains NF-κB in an inactive state by sequestering it in the cytoplasm.
TLR3 functions as a receptor for double-stranded (ds)RNA typically associated with viral infection (4). It was originally shown to be specifically expressed in dendritic cells of the leukocyte family (5). TLR3 has also been found in placenta and lung, and can be induced by LPS in a variety of tissues (4,6). TLR3 is predominantly localized to early endosomes (7,8). Binding of dsRNA, or the analog polyinosine-polycytidylic acid (pIpC), to TLR3 triggers activation of transcription factors NF-κB and IRF3 through the adaptor protein TICAM-1/TRIF (9,10). TRIF associates with members of the TRAF family and with RIP that combine to activate NF-κB and IRF3 (11-13).
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