Western blot analysis of extracts from various cell lines using eIF5 (D5G9) Rabbit mAb.
Western blot analysis of extracts from Huh7 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), eIF5 siRNA I (+), or eIF5 siRNA II (+), using eIF5 (D5G9) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The eIF5 (D5G9) Rabbit mAb confirms silencing of eIF5 expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.
|REACTIVITY||H M R Mk|
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
eIF5 (D5G9) Rabbit mAb recognizes endogenous levels of total eIF5 protein.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 eIF5 protein.
Eukaryotic translation initiation factor 5 (eIF5) is crucial for the assembly of translation initiation complex and plays an important role in protein synthesis (1). eIF5 interacts with the 43S initiation complex to stimulate hydrolysis of GTP bound to eIF2 (1-3). Studies suggest that eIF5 functions as the GTPase-activating protein (GAP) in the hydrolysis of GTP-bound eIF2 (4,5). This hydrolysis leads to the release of initiation factors from the 40S ribosomal subunit, which is a necessary step in the formation of the 80S initiation complex (1).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.