Anti-EPC1 antibody (ab5514)

Immunofluorescence - EPC1 antibody (ab5514)

Specificity of ab5514 against EPC1

A and B: Detection using indirest immunofluorescence of the signal corresponding to EPC1 in EPC1 in 293 Cells were fixed using 4% formaldehyde blocked with PBS containing 3% milk and 0.5% Triton x-100 (sigma) incubated for 1 hour at 37 C with ab5514 at a 1/50 dilution. The cells were then washed three times and incubated for 1 hour at 37 C with a donkey anti-rabbit antibody coupled with Alexa Flour 488 (Molecular probes) at a 1/500 dilution, washed three times, stained with DAPI and mounted with Vectasheild(Abcyss). A: Acquisition on a CCD camera B: Confocal microscopy (3um stack along Z axis).

C. Detection using indirect immunofluoresence of the signals corresponding to the staining with the anti-FLAG mouse antibody (left, red) and the Abcam ab5514 directed against EPC1 (middle, green). 29GT cells were fixed 24 hours after their transfection with a vector containing a FLAG form of EPC1 sequence, using Lipofectamine 2000 transfection procedure (Invitrogen). Mouse anti-FLAG antibody was used at a 1/500 dilution and was detected with donkey anti-mouse antibody coupled with  Alexa Flour 488 (Molecular probes) at a 1/500 dilution. Ab5514 was detected using a donkey anti-rabbit antibody coupled with Alexa Flour 488 (Molecular probes) at a 1/500 dilution. Confocal microscopy.

The endogenous EPC1 protein looks both cytoplasmic and nuclear in 293T cells, which is suprising. However, the few cells that survive the overexpression of a flagged form of the protein display an essentially nuclear  Flag signal.

- EPC1 antibody (ab5514)

Verification of the specificity of ab5514 against EPC1

D: Detection using indirect immunofluorescence of the signal corresponding to EPC1 in 293 cells. Cells were transfected with a control shRNA directed against luciferase (Siluc, left) or two siRNAs directed againstEPC1 (siR1EPC1, middle, and siR2EPC1, right). 24 hours after transfection cells were fixed, blocked, incubated for 1 hr at 37 C with ab5514 at a 1/50 dilution. The cells were then washed three times and incubated for 1 hour at 37 C with a donkey anti-rabbit antibody coupled with Alexa Flour 488 (Molecular probes) at a 1/500 dilution, washed three times, stained with DAPI and mounted with Vectasheild(Abcyss). E: Efficiency of expression knock-down by the SiRNAs against EPC1. Transfection was down as described in D. Quantification of EPC1 mRNA level, expressed as a ratio to RPL19 mRNA and compared to the control (siRI Luc transfection), using quantitative RT-PCR.

Genevive Fourel

Western blot - EPC1 antibody (ab5514)

All lanes : Anti-EPC1 antibody (ab5514) at 2 µg/ml

Lane 1 : Whole cell lysate from HEK293 cell line
Lane 2 : Whole cell lysate from HeLa cell line
Lane 3 : Whole cell lysate from U20S cell line

Lysates/proteins at 30 µg per lane.

Secondary
HRP-conjugated pig anti rabbit Ig

Performed under reducing conditions.

Exposure time : 15 minutes

Detection method: Supersignal West Dura. Note: There are 3 characterised isoforms of EPC1 which give rise to proteins of 93 kDa, 90 kDa and 85 kDa (Swissprot). This antibody detects a species of ~85 kDa in all lysates, including HEK293 cells which are known to express EPC1. The clean blots, together with the IF data (see datasheet), suggests that this antibody is specific to EPC1 and will work in western blotting.

This image is courtesy of an Abreview submitted by Antibody Solutions Ltd.