Anti-Clusterin antibody [EPR2911] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

This data was developed using ab92548, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling CLU with ab92548 at 0.1ug/ml followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining in human Alzheimer's brain.

The section was incubated with ab92548 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

ab92548 (unpurified) at 1/100 dilution staining Apolipoprotein J in Human testis tissue by Immunohistochemistry Formalin-fixed, Paraffin-embedded tissue.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92548).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling Clusterin with purified ab92548 at 1/450 dilution (0.23 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92548).

• WB

Lab

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

This data was developed using ab92548, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[EPR2911] to Clusterin ab92548 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 60 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in CLU knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Clusterin antibody [EPR2911] (<a href='/en-us/products/primary-antibodies/clusterin-antibody-epr2911-ab92548'>ab92548</a>) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Human CLU knockout U-87 MG cell line at 20 µg

Lane 3:

Wild-type HAP1 at 20 µg

Lane 4:

CLU knockout HAP1 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

false

• WB

Unknown

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92548).

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Clusterin knockout HAP1 whole cell lysate (20 μg)
Lane 3 : U87-MG whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab92548 observed at 68 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab92548 was shown to recognize Clusterin in wild-type HAP1 cells as signal was lost at the expected MW in Clusterin knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Clusterin knockout samples were subjected to SDS-PAGE. ab92548 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (ab271867)

Predicted band size: 52 kDa

Observed band size: 68 kDa

false

• WB

Unknown

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92548).

Clusterin precursor could be cleaved to produce an N-terminal α-chain and a C-terminal β-chain as was described by PMID : 25402950. ab92548 recognizes α-chain.

All lanes:

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (ab271867) at 1/1000 dilution

Lane 1:

Human testis lysates at 20 µg

Lane 2:

Human brain lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 52 kDa

Observed band size: 37 kDa,68 kDa

false

• WB

Unknown

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (AB271867)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92548).

All lanes:

Western blot - Anti-Clusterin antibody [EPR2911] - BSA and Azide free (ab271867) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 10 µg

Lane 2:

Fetal brain tissue lysate at 10 µg

Lane 3:

Human Plasma lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 52 kDa

false