All lanes : Anti-Lin28B antibody [EPR18717] (ab191881) at 1/2000 dilution

Lane 1 : Wild-type HEK293 cell lysate
Lane 2 : LIN28B knockout HEK293 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 27 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab191881 observed at 35 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab191881 was shown to react with Lin28B in wild-type HEK-293 cells in Western blot with loss of signal observed in LIN28B knockout sample. Wild-type HEK-293 and LIN28B knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab191881 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 2000 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.

ab191881 staining Lin28B in wild-type HEK293 cells (top panel) and Lin28B knockout HEK293 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab191881 at 10µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).

All lanes : Anti-Lin28B antibody [EPR18717] (ab191881) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : LIN28B knockout HEK293T cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : SW480 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 27 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab191881 observed at 36 kDa. Red - loading control ab7291 observed at 50 kDa.

ab191881 Anti-Lin28B antibody [EPR18717] was shown to specifically react with Lin28B in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab265066 (knockout cell lysate ab257504) was used. Wild-type and Lin28B knockout samples were subjected to SDS-PAGE. ab191881 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C 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 20000 dilution for 1 hour at room temperature before imaging.

 

Anti-Lin28B antibody [EPR18717] (ab191881) at 1/2000 dilution + Recombinant full length Human Lin28B protein at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 27 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Recombinant Human Lin28B protein contains aa1-250 with His-Tag®.

Anti-Lin28B antibody [EPR18717] (ab191881) at 1/2000 dilution + Human placenta lysate at 20 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 27 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Lin28B antibody [EPR18717] (ab191881) at 1/2000 dilution + Human testis lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 27 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Lin28B antibody [EPR18717] (ab191881) at 1/2000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 27 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Lin28B antibody [EPR18717] (ab191881) at 1/2000 dilution + NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 27 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized JAR (Human placenta choriocarcinoma cell line) cells labeling Lin28B with ab191881 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasm and nuclear staining on JAR cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab191881 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling Lin28B with ab191881 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasm and nuclear staining on K562 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab191881 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling Lin28B with ab191881 at 1/150 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

Lin28B was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with ab191881 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab191881 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: K562 whole cell lysate 10ug (Input).

Lane 2: ab191881 IP in K562 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab191881 in K562 whole cell lysate.

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

Exposure time: 5 seconds.