711273

antibody from Invitrogen Antibodies

Targeting: YWHAE FLJ45465

Western blot (Supportive data in Antibodypedia)

Antibody Data

Product number

711273

Provider

Invitrogen Antibodies

Product name

14-3-3 epsilon Recombinant Polyclonal Antibody (5HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Rat

Host

Rabbit

Isotype

IgG

Antibody clone number

5HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2) and Membrane extract of Jurkat (Lane 3), PC-12 (Lane 4), SW480 (Lane 5), NIH/3T3 (Lane 6), Hep G2 (Lane 7), HeLa (Lane 8) and T-47D (Lane 9). The blots were probed with Anti-14-3-3 protein epsilon/YWHAE Recombinant Rabbit Polyclonal Antibody (Product # 711273, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 29 kDa band corresponding to 14-3-3 protein epsilon/YWHAE was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of 14-3-3 epsilon was achieved by transfecting SH-SY5Y with 14-3-3 epsilon specific siRNAs (Silencer® select Product # s200463). Western blot analysis (Fig. a) was performed using modified whole cell extracts from the 14-3-3 epsilon knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with 14-3-3 epsilon Antibody (5HCLC), ABfinity Rabbit Oligoclonal (Product # 711273, 0.25ug/ml ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to 14-3-3 epsilon upon transfecting SH-SY5Y cells with 14-3-3 epsilon specific siRNAs.