Recombinant Anti-CGRP-I+CGRP-II antibody [EPR23804-95] (ab283568)

Immunohistochemical analysis of paraffin-embedded Mouse spinal cord tissue labelling CGRP-I+CGRP-II with ab283568 at 1/1000 (0.639 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse central termination sensory neurons (PMID: 29634489). The section was incubated with ab283568 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat spinal cord tissue labelling CGRP-I+CGRP-II with ab283568 at 1/1000 (0.639 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat central termination sensory neurons. The section was incubated with ab283568 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labelling CGRP-I+CGRP-II with ab283568 at 1/1000 (0.639 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the muscularis nerve fibres of mouse colon. The section was incubated with ab283568 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse skin tissue labelling CGRP-I+CGRP-II with ab283568 at 1/1000 (0.639 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The section was incubated with ab283568 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Negative control: no staining on mouse skin.

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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat skin tissue labelling CGRP-I+CGRP-II with ab283568 at 1/1000 (0.639 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The section was incubated with ab283568 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Negative control: no staining on rat skin.

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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spinal cord tissue labeling CGRP-I+CGRP-II with ab283568 at 1/100 (6.39 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution (Green). Positive staining on the dorsal horn of mouse spinal cord is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat spinal cord tissue labeling CGRP-I+CGRP-II with ab283568 at 1/100 (6.39 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution (Green). Positive staining on the dorsal horn of rat spinal cord is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse skin tissue labeling CGRP-I+CGRP-II with ab283568 at 1/100 (6.39 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution (Green).  The nuclear counterstain was DAPI (Blue).

Negative control: No staining on mouse skin is observed.

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat skin tissue labeling CGRP-I+CGRP-II with ab283568 at 1/100 (6.39 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution (Green). The nuclear counterstain was DAPI (Blue).

Negative control: No staining on rat skin is observed.

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized TT cells labelling CGRP-I+CGRP-II with ab283568 at 1/100 (6.39 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing cytoplasmic staining in TT cell line, and no staining in 293T cell line is observed.

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell) (Left) / TT (Human thyroid carcinoma epithelial cell) (Right) cells labelling CGRP-I+CGRP-II with ab283568 at 1/5000 dilution (0.01ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, ab98507) at 1/500 dilution was used as the secondary antibody.

Negative control: 293T.

All lanes : Anti-CGRP-I+CGRP-II antibody [EPR23804-95] (ab283568) at 1/1000 dilution

Lane 1 : TT (human thyroid carcinoma epithelial cell), whole cell lysate
Lane 2 : 293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : Daudi (human Burkitt's lymphoma lymphoblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 10 kDa why is the actual band size different from the predicted?

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

Fresh lysate of TT cell line was used in this blot.

Negative control: 293T, Hela, Daudi  (HPA database)

Exposure time: 26 seconds

 

All lanes : Anti-CGRP-I+CGRP-II antibody [EPR23804-95] (ab283568) at 1/1000 dilution

Lane 1 : His-tagged mouse CALCB recombinant protein
Lane 2 : Mouse CALCA recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 14 kDa why is the actual band size different from the predicted?

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

This antibody has cross-reaction with mouse CALCB.

The rec proteins CALCB and CLACA were made in-house.

CALCB recombinant protein was made in-house and expressed from the E.coli expression system.

CLACA  recombinant protein was expressed from a mammalian - HEK-293 expression system.

Exposure time: 1 second