Calreticulin Antibody - BSA Free

Immunofluorescence Procedure

1) Cell cultures were treated using an HIER [heat-induced epitope retrieval] protocol.
2) Calreticulin polyclonal antibody [NB 600-101] was diluted 1:50 with a DAKO antibody diluent
3) Cultures and primary antibody were incubated at RT for 30 minutes.
4) Cultures were incubated with an Alexa green fluorescent secondary, as per manufacturer'??s protocol.
5) Cultures were observed on a Zeiss Axioconvert microscope and with DigiPix software.

**NOTE: COS7 and HCT29 cell cultures were used as positive controls for this antibody.

Immunohistochemistry Procedure

1) Paraffin-embedded sections were treated using an HIER [heat-induced epitope retrieval] protocol.
2) NB 600-101 was diluted 1:50 with a DAKO antibody diluent
3) Sections and primary antibody were incubated at RT for 30 minutes.
4) Sections were incubated with a DAKO secondary, as per manufacturer'??s protocol.
5) Sections were then stained with DAB and H&E.
6) Sections were mounted with Faramount solution.
7) Result of staining was strong.

**NOTE: normal colon mucosa and adenocarcinoma tissues were used as positive controls for this antibody.

Western Blot Procedure

1) Scrape cells* off culture dishes and centrifuge.
2) Dissolve cell pellet in decanoyl-N-methyl glucamide (MEGA-10)** and clarify by centrifugation.
3) Mix 30 mg of protein*** with sample buffer containing mercaptoethanol and SDS and run on a 10% SDS gel. The protein was electroblotted on to nitrocellulose.
4) Block nitrocellulose with 5% powdered milk in PBS for 1 hour.
5) Wash the blot with PBS.
6) Add the antibody at a concentration of 1:1000 in 5% powdered milk/PBS and incubate for 1 hour.
7) Wash 3 x 5 minutes with PBS.
8) Add peroxidase-labelled anti-rabbit second antibody in PBS at a concentration of 1:3000 and shake for 1 hour.
9) Wash extensively with PBS.
10) Develop with ECL reagents (Amersham). For this experiment, the film was exposed to the blot for 10 seconds. ****

i. The cells used were the H4IIE rat hepatoma cell line and the NBL-1 bovine renal epithelial cell line.
ii. The detergent used is not critical. MEGA-10 has the advantage of not interfering with the Bradford protein reagent.
iii. Whole cells were used in this experiment. If 30mg of a cell membrane fraction were used a more intense band would be seen.
iv. In this experiment, the antibody was used at 1:1000, but since whole cell protein was used and only 10 seconds development was required it could presumably be used at a lower concentration for many applications.