- 710627 - Invitrogen Antibodies CYCS antibody

Li L, Thompson J, Hu Y, Lesnefsky EJ, Willard B, Chen Q
Scientific reports 2022 Jan 7;12(1):138

Chen Q, Thompson J, Hu Y, Lesnefsky EJ
Free radical biology & medicine 2020 Oct;158:162-170

Chen Q, Thompson J, Hu Y, Dean J, Lesnefsky EJ
American journal of physiology. Cell physiology 2019 Nov 1;317(5):C910-C921

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Cytochrome c was performed by loading 20 µg of HeLa, MDA-MB-231, HEK-293, U-87 MG and Hep G2 cell lysates using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. Cytochrome c was detected at ~16 kDa using Cytochrome c Recombinant Rabbit Polyclonal Antibody (Product # 710627) at 1:1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Cytochrome C was achieved by transfecting HepG2 cells with Cytochrome C specific siRNAs (Silencer® select Product # s28897, s28898). Western blot analysis (Fig. a) was performed using whole cell extracts from Cytochrome C knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Cytochrome C (4HCLC), Recombinant Rabbit Polyclonal Antibody (Product # 710627, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig. b). Reduction of signal upon siRNA mediated knock down confirms that antibody is specific to Cytochrome C.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Cytochrome c was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Cytochrome c Recombinant Rabbit Polyclonal Antibody (Product # 710627) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). F-actin (Panel b: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Nuclei (Panel c: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and panel d is a merged image showing cytoplasmic and perinuclear localization. The images were captured using a Nikon microscope at 20X magnification.

710627