Mouse CD68 knockout RAW 264.7 cell lysate

• WB

Supplier Data

Western blot - Mouse CD68 knockout RAW 264.7 cell lysate (AB280106)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-2 : Merged signal (red and green). Green - ab283654 observed at 100 kDa. Red - loading control ab8245 observed at 36 kDa.
ab283654 Anti-CD68 antibody [EPR23917-164] was shown to specifically react with CD68 in wild-type RAW264.7 cells. Loss of signal was observed when knockout cell line (knockout cell lysate - ab280106) was used. Wild-type and CD68 knockout samples were subjected to SDS-PAGE. ab283654 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4℃ overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD68 antibody [EPR23917-164] (<a href='/en-us/products/primary-antibodies/cd68-antibody-epr23917-164-ab283654'>ab283654</a>) at 1/1000 dilution

Lane 1:

Wild-type RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

Western blot - Mouse CD68 knockout RAW 264.7 cell lysate (ab280106) at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 37 kDa

Observed band size: 100 kDa

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• WB

Lab

Western blot - Mouse CD68 knockout RAW 264.7 cell lysate (AB280106)

Lane 1 : Wild-type RAW 264.7 cell lysate 20 μg

Lane 2 : CD68 knockout RAW 264.7 cell lysate 20 μg

Lane 3 : Mouse spleen cell lysate 20 μg

Lane 4 : Neuro-2a cell lysate 20 μg

False colour image of Western blot : Anti-CD68 antibody staining at 0.2 μg/ml, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab125212 was shown to bind specifically to CD68. A band was observed at 95/102 kDa in wild-type RAW 264.7 cell lysates with no signal observed at this size in CD68 knockout cell line ab280047 (knockout cell lysate ab280106). To generate this image, wild-type and CD68 knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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-CD68 antibody (<a href='/en-us/products/primary-antibodies/cd68-antibody-ab125212'>ab125212</a>) at 0.2 µg/mL

Lane 1:

Wild-type RAW 264.7 cell lysate at 20 µg

Lane 2:

CD68 knockout RAW 264.7 cell lysate at 20 µg

Lane 2:

Western blot - Mouse CD68 knockout RAW 264.7 cell line (<a href='/en-us/products/cell-lines/mouse-cd68-knockout-raw-2647-cell-line-ab280047'>ab280047</a>)

Lane 3:

Mouse spleen cell lysate at 20 µg

Lane 4:

Neuro-2a cell lysate at 20 µg

Predicted band size: 37 kDa

Observed band size: 95-102 kDa

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• Sanger seq

Lab

Sanger Sequencing - Mouse CD68 knockout RAW 264.7 cell lysate (AB280106)

Mouse Cd68 KO in Raw264.7 Cells with 28 bp insertion-13 bp deletion-135 bp insertion-72 bp deletion in Exon 2.