Anti-Factor H antibody [EPR6225]

• WB

Lab

Western blot - Anti-Factor H antibody [EPR6225] (AB133536)

Lanes 1-3 : Merged signal (red and green). Green - ab133536 observed at 180 kDa. Red - loading control ab8245 observed at 36 kDa.

ab133536 Anti-Factor H antibody [EPR6225] was shown to specifically react with Factor H in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267031 (knockout cell lysate ab257150) was used. Wild-type and Factor H knockout samples were subjected to SDS-PAGE. ab133536 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.

All lanes:

Western blot - Anti-Factor H antibody [EPR6225] (ab133536) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

CFH knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CFH (Factor H) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-cfh-factor-h-knockout-a549-cell-line-ab267031'>ab267031</a>)

Lane 3:

HaCaT 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: 139 kDa

Observed band size: 180 kDa

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• WB

Lab

Western blot - Anti-Factor H antibody [EPR6225] (AB133536)

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab133536 overnight at 4°C at a 1/1000 dilution. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Factor H antibody [EPR6225] (ab133536) at 1/1000 dilution

Lane 1:

Purified Factor H protein at 0.5 µg

Lane 2:

Purified Factor H protein at 0.1 µg

Predicted band size: 139 kDa

Observed band size: 170 kDa

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• WB

Unknown

Western blot - Anti-Factor H antibody [EPR6225] (AB133536)

All lanes:

Western blot - Anti-Factor H antibody [EPR6225] (ab133536) at 1/1000 dilution

Lane 1:

Human plasma lysate at 10 µg

Lane 2:

Human kidney lysate at 10 µg

Lane 3:

Human fetal lung lysate at 10 µg

Lane 4:

Human fetal liver lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 139 kDa

Observed band size: 180 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Factor H antibody [EPR6225] (AB133536)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.