Anti-ABCA1 antibody [HJ1] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] - BSA and Azide free (AB264550)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab264550).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] - BSA and Azide free (AB264550)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab264550).

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [HJ1] - BSA and Azide free (AB264550)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] (<a href='/en-us/products/primary-antibodies/abca1-antibody-hj1-ab66217'>ab66217</a>) 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

Supplier Data

Western blot - Anti-ABCA1 antibody [HJ1] - BSA and Azide free (AB264550)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] (<a href='/en-us/products/primary-antibodies/abca1-antibody-hj1-ab66217'>ab66217</a>) 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