- MA5-25697 - Invitrogen Antibodies ACSS2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ACSS2 in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with ACSS2 (Product # MA5-25697) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ACSS2 in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with ACSS2 (Product # MA5-25697) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ACSS2 in HepG2, HeLa, SVT2, A549, COS7, Jurkat, MDCK, PC12 and MCF7 cells using 35 µg per lane. Samples were separated by SDS-PAGE and probed with ACSS2 (Product # MA5-25697) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Acetyl-coenzyme A synthetase, cytoplasmic was achieved by transfecting Hep G2 with Acetyl-coenzyme A synthetase, cytoplasmic specific siRNAs (Silencer® select Product # S31745, S31746). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Acetyl-coenzyme A synthetase, cytoplasmic knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ACSS2 Monoclonal Antibody (OTI3H4) (Product # MA5-25697, 1:2000 dilution ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 Acetyl-coenzyme A synthetase, cytoplasmic.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-ACSS2 Monoclonal Antibody (OTI3H4) (Product # MA5-25697) and a ~75kDa band corresponding to Acetyl-coenzyme A synthetase, cytoplasmic was observed across cell lines tested along with two uncharacterized bands (*) at ~50 kDa and ~80 kDa. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), Hep G2 (Lane 3), PC-3 (Lane 4), A-431 (Lane 5), U-87 MG (Lane 6), PANC-1 (Lane 7), NIH/3T3 (Lane 8) and PC-12 (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Acetyl-coenzyme A synthetase, cytoplasmic 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 5 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with ACSS2 Monoclonal Antibody (OTI3H4) (Product # MA5-25697) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nucleus and cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on paraffin-embedded human pancreas tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a ACSS2 monoclonal antibody (Product # MA5-25697).

MA5-25697