All lanes : Anti-BAF57/SMARCE1 antibody [EPR8848] (ab131328) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SMARCE1 knockout HeLa cell lysate
Lane 3 : MCF7 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: 47 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab131328 observed at 51 kDa. Red - loading control ab8245 observed at 36 kDa.

ab131328 Anti-BAF57/SMARCE1 antibody [EPR8848] was shown to specifically react with BAF57/SMARCE1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265780 (knockout cell lysate ab258200) was used. Wild-type and BAF57/SMARCE1 knockout samples were subjected to SDS-PAGE. ab131328 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.

 

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling BAF57/SMARCE1 with ab131328 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-BAF57/SMARCE1 antibody [EPR8848] (ab131328) at 1/1000 dilution

Lane 1 : MCF-7 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 47 kDa

Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab131328 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemical analysis of paraffin-embedded Human skin tissue labelling BAF57/SMARCE1 with ab131328 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ICC/IF image of ab131328 stained HepG2 cells. The cells were 4% formaldehyde 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 (ab131328, 1/100 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) 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.

Equilibrium disassociation constant (K_D)
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