Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free (AB275933)

This data was developed using ab275931, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling C5/C5b with ab275931 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on plasma of human breast. The section was incubated with ab275931 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free (AB275933)

This data was developed using ab275931, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling C5/C5b with ab275931 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human liver. The section was incubated with ab275931 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Lab

Western blot - Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free (AB275933)

False colour image of Western blot : Anti-C5 / C5b antibody [EPR23940-3] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab275931 was shown to bind specifically to C5 / C5b. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-C5 / C5b antibody [EPR23940-3] (<a href='/en-us/products/primary-antibodies/c5-c5b-antibody-epr23940-3-ab275931'>ab275931</a>) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 20 µg

Lane 2:

U-2 OS cell lysate at 20 µg

Predicted band size: 188 kDa

Observed band size: 165-180 kDa

false

• WB

Lab

Western blot - Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free (AB275933)

This data was developed using ab275931, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregates.

The molecular weight observed is consistent with what has been described in the literature (PMID : 24071736, 23401592, 25387724)

Exposure time : 59 seconds.

All lanes:

Western blot - Anti-C5 / C5b antibody [EPR23940-3] (<a href='/en-us/products/primary-antibodies/c5-c5b-antibody-epr23940-3-ab275931'>ab275931</a>) at 1/1000 dilution

Lane 1:

Human plasma at 10 µg

Lane 2:

Human liver tissue lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 188 kDa

Observed band size: 190 kDa,70 kDa

false

• WB

Lab

Western blot - Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free (AB275933)

This data was developed using ab275931, the same antibody clone in a different buffer formulation.

Western blot : Rabbit monoclonal [EPR23940-3] to C5 / C5b ab275931 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 180 kDa in Wild-type A549 ab288558 cell lysates with no signal observed at this size in C5 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-C5 / C5b antibody [EPR23940-3] (<a href='/en-us/products/primary-antibodies/c5-c5b-antibody-epr23940-3-ab275931'>ab275931</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 ab288558 at 20 µg

Lane 2:

C5 knockout A549 at 20 µg

Lane 3:

HepG2 at 20 µg

Lane 4:

U-2 OS at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 188 kDa

Observed band size: 180 kDa

false

• WB

Lab

Western blot - Anti-C5 / C5b antibody [EPR23940-3] - BSA and Azide free (AB275933)

This data was developed using ab275931, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 23401592, 25387724).

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-C5 / C5b antibody [EPR23940-3] (<a href='/en-us/products/primary-antibodies/c5-c5b-antibody-epr23940-3-ab275931'>ab275931</a>) at 1/1000 dilution

Lane 1:

Mouse plasma at 20 µg

Lane 2:

Rat plasma at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 188 kDa

Observed band size: 70 kDa

false