Anti-Bcl6 antibody [EP529Y] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-Bcl6 antibody [EP529Y] - BSA and Azide free (AB240925)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33901). Bcl6 was immunoprecipitated from Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate with ab33901 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab33901 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution. Lane 1 (Input) : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate, 10 μg Lane 2 (+) : Ramos whole cell lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab33901 in Ramos whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Bcl6 antibody [EP529Y] (<a href='/en-us/products/primary-antibodies/bcl6-antibody-ep529y-ab33901'>ab33901</a>) at 1/1000 dilution

Lane 1:

Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 10 µg

Lane 2:

Ramos whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 79 kDa

Observed band size: 78 kDa

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• WB

Lab

Western blot - Anti-Bcl6 antibody [EP529Y] - BSA and Azide free (AB240925)

This data was developed using the same antibody clone in a different buffer formulation (ab33901).

Lanes 1-4 : Merged signal (red and green). Green ab33901 observed at 78 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab33901 Anti-Bcl6 antibody [EP529Y] was shown to react with BCL6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265410 (knockout cell lysate ab257178) was used. Wild-type and Bcl6 knockout samples were subjected to SDS-PAGE. ab33901 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Bcl6 antibody [EP529Y] (<a href='/en-us/products/primary-antibodies/bcl6-antibody-ep529y-ab33901'>ab33901</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BCL6 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human BCL6 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-bcl6-knockout-hela-cell-line-ab265410'>ab265410</a>)

Lane 3:

Ramos cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 79 kDa

Observed band size: 78 kDa

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• WB

Supplier Data

Western blot - Anti-Bcl6 antibody [EP529Y] - BSA and Azide free (AB240925)

Blocking and diluting buffer : 5% NFDM/TBST.

This antibody works better in 1%SDS Hot Lysates in WB. For Lysate preparation protocol, please refer to the protocol section of the website and/or here.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33901).

All lanes:

Western blot - Anti-Bcl6 antibody [EP529Y] (<a href='/en-us/products/primary-antibodies/bcl6-antibody-ep529y-ab33901'>ab33901</a>) at 1/1000 dilution

Lane 1:

Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysates prepared in 1%SDS Hot lysis method at 15 µg

Lane 2:

Ramos (Human Burkitt's lymphoma lymphoblast) whole cell lysates prepared in 1%SDS Hot lysis method at 15 µg

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 1/20000 dilution

Predicted band size: 79 kDa

false

Exposure time: 10s