Anti-MCP3 antibody [EPR22649-155]

• IP

Unknown

Immunoprecipitation - Anti-MCP3 antibody [EPR22649-155] (AB228979)

MCP3 was immunoprecipitated from 0.35 mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) (treated with 80 nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then with 100 ng/ml lipopolysaccharides (LPS) treated for 6 hours with addition of 1ug/ml of Brefeldin A for the last 3 hours), whole cell lysate with ab228979 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab228979 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1 : RAW264.7 (treated as above) whole cell lysate 10ug

Lane 2 : ab228979 IP in RAW264.7 (treated as above) whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab228979 in RAW264.7 (treated as above) whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 min.

All lanes:

Immunoprecipitation - Anti-MCP3 antibody [EPR22649-155] (ab228979)

Predicted band size: 11 kDa

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• WB

Lab

Western blot - Anti-MCP3 antibody [EPR22649-155] (AB228979)

Lanes 1 - 6 : Merged signal (red and green). Green - ab228979 observed at 14 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab228979 was shown to react with MCP3 in RAW 264.7 wild-type cells in Western blot with loss of signal observed in CCL7 knockout sample. Wild-type and CCL7 knockout RAW 264.7 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab228979 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-MCP3 antibody [EPR22649-155] (ab228979) at 1/1000 dilution

Lane 1:

Wild-type RAW 264.7 untreated control cell lysate at 30 µg

Lane 2:

Wild-type RAW 264.7 PMA treated (80 nM, 24 h) plus LPS treated (100 ng/ml, 6 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 5 h) cell lysate at 30 µg

Lane 2:

Western blot - Mouse CCL7 knockout RAW 264.7 cell line (<a href='/en-us/products/cell-lines/mouse-ccl7-knockout-raw-2647-cell-line-ab273755'>ab273755</a>)

Lane 3:

CCL7/MCP3 knockout RAW 264.7 untreated cell lysate at 30 µg

Lane 4:

CCL7/MCP3 knockout RAW 264.7 PMA treated (80 nM, 24 h) plus LPS treated (100 ng/ml, 6 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 5 h) cell lysate at 30 µg

Lane 5:

J774A.1 Glucose treated (138.8 mMol/L, 8 h) plus Brefeldin A treated (5 µg/ml, 6 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 5 h) cell lysate at 30 µg

Lane 6:

J774A.1 Glucose treated (5.6 mMol/L, 8 h) plus Brefeldin A treated (5 µg/ml, 6 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 5 h) cell lysate at 30 µg

Predicted band size: 11 kDa

Observed band size: 14 kDa

false

• WB

Lab

Western blot - Anti-MCP3 antibody [EPR22649-155] (AB228979)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-MCP3 antibody [EPR22649-155] (ab228979) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 40 µg

Lane 2:

RAW264.7 (treated with 80 nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then with 100 ng/ml lipopolysaccharides (LPS) treated for 6 hours with addition of 1ug/ml of Brefeldin A for the last 3 hours), whole cell lysate 40 ug.

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 1/100000 dilution

Predicted band size: 11 kDa

Observed band size: 11 kDa

false

• WB

Lab

Western blot - Anti-MCP3 antibody [EPR22649-155] (AB228979)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Western blot - Anti-MCP3 antibody [EPR22649-155] (ab228979) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

HEK-293T transfected with MCP3 expression vector containing a myc-His-tag®, whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 11 kDa

Observed band size: 16-25 kDa

false