Western blot analysis of extracts from 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® GABARAPL2 siRNA I # 14239 (+) or SignalSilence® II GABARAPL2 siRNA II #14246 (+), using GABARAPL2 (D1W9T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The GABARAPL2 (D1W9T) Rabbit mAb confirms silencing of GABARAPL2, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
Western blot analysis of extracts from various cell lines using GABARAPL2 (D1W9T) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human GABARAP (hGABARAP-Myc/DDK; +), human GABARAPL1 (hGABARAPL1-Myc/DDK; +), or human GABARAPL2 (hGABARAPL2-Myc/DDK; +), using GABARAPL2 (D1W9T) Rabbit mAb.
Western blot analysis of extracts from A172 and HeLa cells, untreated (-) or treated overnight with chloroquine (50 μM) (+), using GABARAPL2 (D1W9T) Rabbit mAb (upper) or β-Actin (D6A8) #8457 (lower).
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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
GABARAPL2 (D1W9T) Rabbit mAb recognizes endogenous levels of total GABARAPL2 protein. Bands of unknown origin are detected at 80 and 110 kDa in some cell lines. This antibody has a preference for the Type I form of GABARAPL2.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human GABARAPL2 protein.
GABAA receptor associated protein (GABARAP) is an Atg8 family protein with a key role in autophagy, which was originally discovered as a protein associated with the GABAA receptor regulating receptor trafficking to the plasma membrane (1). Proteins in this family, including microtubule-associated protein light chain 3 (LC3) and GATE-16 (GABARAPL2), become incorporated into the autophagosomal membranes following autophagic stimuli such as starvation (2). Like the other family members, GABARAP is cleaved at its carboxyl terminus, which leads to conjugation by either of the phospholipids phosphatidylethanolamine or phosphatidylserine (3,4). This processing converts GABARAP from a type I to a type II membrane bound form involved in autophagosome biogenesis. Processing of GABARAP involves cleavage by Atg4 family members (5,6) followed by conjugation by the E1 and E2 like enzymes Atg7 and Atg3 (7,8). GABARAPL1/GEC1, a protein that is highly related to GABARAP, was identified as an estrogen inducible gene, and is also associated with autophagosomes (9-11).
GABARAPL2/GATE-16 was identified as a modulator of membrane transport, interacting with N-ethylmaleimide senstive factor (NSF) and the Golgi v-SNARE GOS-28 (12). In addition, GABARAPL2 interacts with OSBP-related protein 7 (ORP7), the GTPase GIMAP6, and the calcium channel TRPML3. (13-15)
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