Anti-Lin28B antibody [EPR25252-20] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Lin28B antibody [EPR25252-20] - BSA and Azide free (AB290003)

This data was developed using ab289978, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Lin28B with ab289978 at 1/500 (1.106 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing positive staining in NIH/3T3 cells transfected with a mouse Lin28B expression vector containing a myc tag. is observed. Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 0.38ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Lin28B antibody [EPR25252-20] - BSA and Azide free (AB290003)

Flow cytometric analysis of 4% paraformaldehyde fixed 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 (0.1ug)/ Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• WB

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Western blot - Anti-Lin28B antibody [EPR25252-20] - BSA and Azide free (AB290003)

This data was developed using ab289978, the same antibody clone in a different buffer formulation.

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.

All lanes:

Western blot - Anti-Lin28B antibody [EPR25252-20] (<a href='/en-us/products/primary-antibodies/lin28b-antibody-epr25252-20-ab289978'>ab289978</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Lin28B knockout HEK293T cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Observed band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-Lin28B antibody [EPR25252-20] - BSA and Azide free (AB290003)

This data was developed using ab289978, the same antibody clone in a different buffer formulation. Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Lin28B antibody [EPR25252-20] (<a href='/en-us/products/primary-antibodies/lin28b-antibody-epr25252-20-ab289978'>ab289978</a>) at 1/1000 dilution

Lane 1:

His-tagged Human Lin-28 homolog B recombinant protein

Lane 2:

His-tagged Human Lin-28 homolog A recombinant protein

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

Observed band size: 35 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Lin28B antibody [EPR25252-20] - BSA and Azide free (AB290003)

This data was developed using ab289978, the same antibody clone in a different buffer formulation. Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Lin28B antibody [EPR25252-20] (<a href='/en-us/products/primary-antibodies/lin28b-antibody-epr25252-20-ab289978'>ab289978</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) transfected with an empty vector (vector control), containi a myc-His-tag®, whole cell lysate 20

Lane 2:

NIH/3T3 cells transfected with a mouse Lin28B expression vector containi a myc-His-tag®, whole cell lysate, 20

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

Observed band size: 35 kDa

false

Exposure time: 5s