Anti-LRP5 antibody [EPR22477-218] - BSA and Azide free

• IP

Unknown

Immunoprecipitation - Anti-LRP5 antibody [EPR22477-218] - BSA and Azide free (AB256528)

LRP5 was immunoprecipitated from 0.35 mg 3T3-L1 (mouse embryonic fibroblast) differentiated adipocytes whole cell lysate 10μg with ab223203 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab223203. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1 : 3T3-L1 (mouse embryonic fibroblast) differentiated adipocytes whole cell lysate 10μg.

Lane 2 : ab223203 IP in 3T3-L1 differentiated adipocytes whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab223203 in 3T3-L1 differentiated adipocytes whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 mins.

Differentiation procedure : https : //www.abcam.com/protocols/differentiation-of-3t3-l1-cells-into-adipocyte-like-cells-protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223203).

All lanes:

Immunoprecipitation - Anti-LRP5 antibody [EPR22477-218] (<a href='/en-us/products/primary-antibodies/lrp5-antibody-epr22477-218-ab223203'>ab223203</a>)

Predicted band size: 179 kDa

Observed band size: 180-200 kDa

false

• WB

Lab

Western blot - Anti-LRP5 antibody [EPR22477-218] - BSA and Azide free (AB256528)

This data was developed using the same antibody clone in a different buffer formulation (ab223203).

Lanes 1-3 : Merged signal (red and green). Green - ab223203 observed at 180-200 kDa. Red - loading control ab7291 observed at 50 kDa.

ab223203 Anti-LRP5 antibody [EPR22477-218] was shown to specifically react with LRP5 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266618 (knockout cell lysate ab257202) was used. Wild-type and LRP5 knockout samples were subjected to SDS-PAGE. ab223203 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 500 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.

All lanes:

Western blot - Anti-LRP5 antibody [EPR22477-218] (<a href='/en-us/products/primary-antibodies/lrp5-antibody-epr22477-218-ab223203'>ab223203</a>) at 1/500 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

LRP5 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human LRP5 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-lrp5-knockout-hek-293t-cell-line-ab266618'>ab266618</a>)

Lane 3:

SW620 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

Predicted band size: 179 kDa

Observed band size: 180-200 kDa

false

• WB

Lab

Western blot - Anti-LRP5 antibody [EPR22477-218] - BSA and Azide free (AB256528)

Blocking and dilution buffer : 5%NFDM/TBST. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223203).

Lanes 1 - 3:

Western blot - Anti-LRP6 (phospho S1490) + LRP5 (phospho S1503) antibody [EP2360Y] (<a href='/en-us/products/primary-antibodies/lrp6-phospho-s1490-lrp5-phospho-s1503-antibody-ep2360y-ab76417'>ab76417</a>) at 1/10000 dilution

Lanes 4 - 6:

Western blot - Anti-LRP5 antibody [EPR22477-218] (<a href='/en-us/products/primary-antibodies/lrp5-antibody-epr22477-218-ab223203'>ab223203</a>) at 1/10000 dilution

Lanes 1 and 4:

293T transfected with blank vector whole cell lysate at 20 µg

Lanes 2 and 5:

293T transfected with LRP6 overexpression vector whole cell lysate at 20 µg

Lanes 3 and 6:

293T transfected with LRP5 overexpression vector whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 179 kDa

Observed band size: 180 kDa

false

Exposure time: 180s