Anti-CD64 antibody [EPR4624] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-CD64 antibody [EPR4624] - BSA and Azide free (AB216857)

This data was developed using ab216857, the same antibody clone in a different buffer formulation.
Purified ab216857 at 1 : 30 dilution (2μg) immunoprecipitating CD64 in THP-1 whole cell lysate.
Lane 1 (input) : THP-1 (Human monocytic leukemia monocyte) whole cell lysate 10μg.
Lane 2 (+) : ab216857 + THP-1 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab134073 in THP-1 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)™(ab131366) (1 : 1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : Glycosylated form : 64-78kDa; Non-glycosylated form : 39-43kDa.

All lanes:

Immunoprecipitation - Anti-CD64 antibody [EPR4624] (<a href='/en-us/products/primary-antibodies/cd64-antibody-epr4624-ab134073'>ab134073</a>)

Predicted band size: 43 kDa

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• WB

Unknown

Western blot - Anti-CD64 antibody [EPR4624] - BSA and Azide free (AB216857)

Glycosylated form : 64-78kDa; Non-glycosylated form : 39-43kDa

All lanes:

Western blot - Anti-CD64 antibody [EPR4624] (<a href='/en-us/products/primary-antibodies/cd64-antibody-epr4624-ab134073'>ab134073</a>) at 1/10000 dilution

Lane 1:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 15 µg

Lane 2:

U-937 (Human histiocytic lymphoma monocyte) whole cell lysate at 15 µg

Secondary

All lanes:

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

Predicted band size: 43 kDa

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• WB

Lab

Western blot - Anti-CD64 antibody [EPR4624] - BSA and Azide free (AB216857)

False colour image of Western blot : Anti-CD64 antibody [EPR4624] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab134073 was shown to bind specifically to CD64. A band was observed at 45-50/55-80 kDa in wild-type THP-1 cell lysates with no signal observed at this size in FCGR1A knockout cell line ab275843 (knockout cell lysate ab275817). To generate this image, wild-type and FCGR1A knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CD64 antibody [EPR4624] (<a href='/en-us/products/primary-antibodies/cd64-antibody-epr4624-ab134073'>ab134073</a>) at 1/10000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

FCGR1A knockout THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human FCGR1A knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-fcgr1a-knockout-thp-1-cell-line-ab275843'>ab275843</a>)

Lane 3:

U937 cell lysate at 20 µg

Lane 4:

HEK-293 cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 45 kDa,45-50 kDa

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