Anti-Lin28B 抗体 [EPR25252-20] (ab289978)

Blocking and diluting buffer and concentration: ½ volume of Odyssey Blocking Buffer (TBS)+ ½ volume of 0.1% TBS.

False colour image of Western blot: Anti-Lin28B antibody EPR25252-20 staining at 1/1000 dilution, shown in green; Mouse anti-Tubulin antibody [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab289978 was shown to bind specifically to Lin28B. The band was observed at 32 kDa in wild-type HEK293T cell lysates with no signal observed at this size in Lin28B knockout cell line ab265066 (knockout cell lysate ab257504) used. To generate this image, wild-type and Lin28B knockout HEK293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 1/10000 dilution.

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

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

This antibody does not cross-react with Human Lin-28 homolog A.
Both recombinant proteins were made in-house and expressed from E.coli expression systems.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblasts) labeling Lin28B with ab289978 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) secondary antibody at 1/1000 dilution (green).

Confocal image showing positive staining in NIH/3T3 cells transfected with a mouse Lin28B expression vector containing a myc tag. A Mouse mAb anti Myc-Tag (Alexa Fluor^® 647) antibody was used as a counterstain (RED). The nuclear counter stain used is DAPI (blue).

The lower panels show secondary only controls where secondary but no primary antibody was added to Myc transfected and LYN28B-Myc transfected cells. DAPI was used as the nuclear counter stain.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells transfected with a mouse Lin28B expression vector containing a myc-His-tag® cells labelling Lin28B with ab289978 at 1/500 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) (Left) isotype control. Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.