Anti-ABCA1 antibody [HJ1]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] (AB66217)

ab66217 (4µg/ml) staining ABCA1 in human liver using an automated system (DAKO Autostainer Plus). Using this protocol there is moderate cell membrane staining throughout the liver parenchyma.

Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] (AB66217)

IHC image of ABCA1 staining in human liver formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab66217, 0.2µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ABCA1 antibody [HJ1] (AB66217)

Flow cytometry overlay histogram showing Hep G2 cells stained with ab66217 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10%; normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab66217) (1x 106 in 100 μl at 5.0 μg/ml (1/0)) for 30 min at 22°C.

The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Mouse IgG2b, kappa monoclonal [7E10G10] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [HJ1] (AB66217)

Blocking and diluting buffer : 5% NFDM/TBST.

ab129002 was used as a loading control at 1/10000 dilution, followed by a secondary antibody ab97051 at 1/20000 dilution.

Negative control : 293T (PMID : 11896206), SH-SY5Y (PMID : 23600914).

Samples are non-boiled as boiling may cause protein aggregates.

All lanes:

Western blot - Anti-ABCA1 antibody [HJ1] (ab66217) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Lanes 1 - 3:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 254 kDa

Observed band size: 254 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [HJ1] (AB66217)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : Empty
Lane 3 : ABCA1 knockout (KO) HAP1 whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab66217 observed at 240 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab66217 detected the expected band for ABCA1 in wild type HAP1 cells and the band was not seen in ABCA1 knockout HAP1 cells, although additional non-specific bands were also seen. Wild-type and ABCA1 knockout samples were subjected to SDS-PAGE. ab66217 and ab176560 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ABCA1 antibody [HJ1] (ab66217)

Predicted band size: 254 kDa

false

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [HJ1] (AB66217)

Blocking and diluting buffer : 5% NFDM/TBST.

ab129002 was used as a loading control at 1/10000 dilution, followed by a secondary antibody ab97051 at 1/20000 dilution.

The higher band is probably glycosylated form (PMID : 16873719).

All lanes:

Western blot - Anti-ABCA1 antibody [HJ1] (ab66217) at 1/1000 dilution

Lane 1:

Untreated THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 treated first with 100ng/ml PAM for 72 hours and then replaced with 100ng/ml LPS for 5 hours, 300ng/ml BFA was then added for additional 3 hours whole cell lysate at 20 µg

Lane 3:

Untreated HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 treated with 20uM T0901317 for 16 hours whole cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Lanes 1 - 3:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 254 kDa

Observed band size: 254 kDa,400 kDa

false

Exposure time: 20s

• WB

Project

Western blot - Anti-ABCA1 antibody [HJ1] (AB66217)

Abcam recommends using milk as the blocking agent. This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab66217 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-ABCA1 antibody [HJ1] (ab66217) at 1 µg/mL

Lane 1:

Brain (Mouse) Tissue Lysate at 20 µg

Lane 2:

Brain (Rat) Tissue Lysate at 20 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 4:

Liver (Mouse) Tissue Lysate at 20 µg

Lane 5:

Liver (Rat) Tissue Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Predicted band size: 254 kDa

Observed band size: 254 kDa,70 kDa

false

Exposure time: 4min