Anti-YB1 antibody (ab12148)

Western blot - YB1 antibody (ab12148)

Anti-YB1 antibody (ab12148) at 1 µg/ml + HEK293 Whole Cell Lysate Transiently Overexpressing YB1 at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 36 kDa
Observed band size : 50 kDa (why is the actual band size different from the predicted?)


YB1 has a predicted band size of 36kDa based on its primary sequence (SwissProt). According to Evdolimova (1995) YB1 migrates by SDS-PAGE at 50kDa, which may be due to post-translational modification

Western blot

All lanes : Anti-YB1 antibody (ab12148) at 1.4 µg/ml

Lane 1 : HeLa Nuclear lysate
Lane 2 : HeLa Whole cell lysate
Lane 3 : MCF-7 cell lysate
Lane 4 : Jurkat whole cell lysate
Lane 5 : HEK293 Whole cell lysate
Lane 6 : HeLa Nuclear lysate with YB1 peptide (ab12411) at 1 µg/ml
Lane 7 : HeLa Whole cell lysate with YB1 peptide (ab12411) at 1 µg/ml
Lane 8 : MCF-7 cell lysate with YB1 peptide (ab12411) at 1 µg/ml
Lane 9 : Jurkat whole cell lysate with YB1 peptide (ab12411) at 1 µg/ml
Lane 10 : HEK293 whole cell lysate with YB1 peptide (ab12411) at 1 µg/ml

Lysates/proteins at 20 µg per lane.


Predicted band size : 36 kDa
Observed band size : 36 kDa
Additional bands at : 50 kDa. We are unsure as to the identity of these extra bands.

A band of approx 50 KDa was partially blocked in several cell lines using a YB1 blocking peptide (ab12411). This suggests the antibody recognises YB1. Another band of approx 36 kDa also appears to be recognised, which may be a cleavage product of YB1.

Immunocytochemistry/ Immunofluorescence - YB1 antibody (ab12148)

ICC/IF image of ab12148 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 5 min) and incubated with the antibody (ab12148, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunocytochemistry/ Immunofluorescence - YB1 antibody (ab12148)

ICC/IF image of ab12148 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 5 min) and incubated with the antibody (ab12148, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - YB1 antibody (ab12148)

Image courtesy of Human Protein Atlas

ab12148 staining YB1 in Human duodenum, showing a distinct and strong staining pattern of the glandular cells. Paraffin embedded human duodenum was incubated with ab12148 (1/5000 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab12148 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

Immunoprecipitation - YB1 antibody (ab12148)

ab12148 was diluted 1/500 and incubated with A2780 whole cell lysate and protein A/G matrix for 16 hours at 4°C to achieve immunoprecipitation. 200 µg of protein was present in the lysate input.  Lane 2 shows results of a different antibody used as the  negative control.  An HRP-conjugated goat anti-rabbit antibody was used for the western blot step.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - YB1 antibody (ab12148)

ICC/IF image of ab12148 stained Hela cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab12148, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.