ab84340 staining golgin-97 in MCF7 cells treated with brefeldin A (ab120299), by ICC/IF. Increase in Golgin-97 expression correlates with increased concentration of brefeldin A, as described in literature.
The cells were incubated at 37°C for 1.5 h in media containing different concentrations of ab120299 (brefeldin A) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab84340 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

All lanes : Anti-MCP3 antibody [EPR22649-155] (ab228979) at 1/1000 dilution

Lane 1 : Wild-type RAW 264.7 untreated control cell lysate
Lane 2 : Wild-type RAW 264.7 PMA treated (80 nM, 24 h) plus LPS treated (100 ng/ml, 6 h) and Brefeldin A (ab120299) treated (5 µg/ml, 5 h) cell lysate
Lane 3 : CCL7/MCP3 knockout RAW 264.7 untreated cell lysate
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 (ab120299) treated (5 µg/ml, 5 h) cell lysate
Lane 5 : J774A.1 Glucose treated (138.8 mMol/L, 8 h) plus Brefeldin A treated (5 µg/ml, 6 h) and Brefeldin A (ab120299) treated (5 µg/ml, 5 h) cell lysate
Lane 6 : J774A.1 Glucose treated (5.6 mMol/L, 8 h) plus Brefeldin A treated (5 µg/ml, 6 h) and Brefeldin A (ab120299) treated (5 µg/ml, 5 h) cell lysate

Lysates/proteins at 30 µg per lane.

Observed band size: 14 kDa why is the actual band size different from the predicted?

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 : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution

Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 3 : TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate

Lysates/proteins at 30 µg per lane.

Observed band size: 26 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab255275 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab255275 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab255275 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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 : Anti-TNF alpha antibody [EPR19147] (ab183218) at 1/1000 dilution

Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 3 : TNF alpha knockout THP-1 Brefeldin A treated (5 µg/ml, 4 h) cell lysate
Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate

Lysates/proteins at 30 µg per lane.

Observed band size: 26 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab183218 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab183218 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab183218 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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 : Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 2 : Wild-type A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 3 : IP10 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 4 : IP10 knockout A549 IFN-y (ab259377) (100ng/ml, 32h) and TNF-alpha (ab259410) (10ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 5 : THP-1 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 6 : THP-1 IFN-y (ab259377) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Observed band size: 11 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab214668 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab214668 was shown to react with IP10 in wild-type A549 cells in western blot with loss of signal observed in IP10 knockout cell line ab266971 (knockout cell lysate ab256888). Wild-type and IP10 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214668 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^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IP10 antibody [EPR7850] (ab137018) at 1/500 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 2 : Wild-type A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml) for 32 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 3 : IP10 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 4 : IP10 knockout A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml) for 32 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Observed band size: 11 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab137018 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab137018 was shown to react with IP10 in A549 wild-type cells in western blot with loss of signal observed in IP10 knockout cell line ab266969 (IP10 knockout cell lysate ab256886). A549 wild-type and IP10 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab137018 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 500 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^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IL-6 antibody [EPR21711] (ab233706) at 1/1000 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 2 : Wild-type A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate
Lane 3 : IL-6 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 4 : IL-6 knockout A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Observed band size: 25 kDa why is the actual band size different from the predicted?
Additional bands at: 40 kDa (possible non-specific binding)

Lanes 1 - 4: Merged signal (red and green). Green - ab233706 observed at 25 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab233706 was shown to react with IL-6 in wild-type A549 cells in western blot with loss of signal observed in IL-6 knockout cell line ab273751 (knockout cell lysate ab275501). Wild-type and IL-6 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab233706 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] 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 hour at room temperature before imaging.

All lanes : Anti-IL-6 antibody [EPR20653] (ab214429) at 1/1000 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 2 : Wild-type A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate
Lane 3 : IL-6 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 4 : IL-6 knockout A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Lanes 1 - 4: Merged signal (red and green). Green - ab214429 observed at 25 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab214429 was shown to react with IL-6 in wild-type A549 cells in western blot with loss of signal observed in IL-6 knockout cell line ab273751 (knockout cell lysate ab275501). Wild-type A549 and IL-6 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214429 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] 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 hour at room temperature before imaging.