PA1-545

antibody from Invitrogen Antibodies

Targeting: PCYT2 ET

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

PA1-545

Provider

Invitrogen Antibodies

Product name

PCYT2 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

0.4 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of ECT was performed using whole cell lysates and tissue lysate of Caco-2 (Lane 1), Hep G2 (Lane 2) and Mouse heart (Lane 3). The blot was probed with Anti-ECT Rabbit polyclonal Antibody (Product # PA1-545, 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 ~ 43 kDa band corresponding to ECT was observed across cell lines and tissue lysate 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® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of PCYT2 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with PCYT2 Polyclonal Antibody (Product # PA1-545) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). GRP94 (Panel c: red) was stained with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Product # A-31572,2 µg/mL). Panel d represents the merged image showing co-localization of PCYT2 with GRP94 in Endoplasmic Reticulum. Panel e represents the control cells showing no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.