MCM (Minichromosome Maintenance Protein) Antibody Staining Protocol for Immunohistochemistry

MCM (pan-Minichromosome Maintenance Protein) Antibody Staining Protocol for Immunohistochemistry

Description: Minichromosome maintenance (MCM) proteins play an essential role for the initiation of eukaryotic DNA replication and bind with chromatin in vivo. They act as factors that ‘license’ DNA for exactly one round of replication per cell cycle. MCM proteins share a highly conserved domain of about 210 amino-acid residues. This domain shares some similarities with that of various other families of DNA-dependent ATPases, which implies that MCM proteins may be involved in an ATP-consuming step in the initiation of DNA replication. Eukaryotes possess a family of six MCM proteins (MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7) that contain this domain. MCM proteins were first identified in yeast where most of them have a direct role in the initiation of chromosomal DNA replication by interacting directly with autonomously replicating sequences (ARS). They were thus called 'minichromosome maintenance proteins' with gene symbols prefixed by MCM. Human MCM2, also called CDCL1, mitotin, D3S3194, KIAA0030, BM28, or cdc19, is a nuclear protein that seems to play an important role in two crucial steps of the cell cycle, namely, onset of DNA replication and cell division. Burkhart et al. showed that human MCM2 and MCM5 form a protein complex.5 Furthermore, DNA synthesis can be blocked by microinjection of antibodies to MCM2. In HeLa cells, MCM protein levels remain relatively constant except for fluctuations during the G0 to G1/S period whereby in G0, MCM 2 and MCM5 are found at much lower concentrations when compared to MCM3 and MCM7. MCM proteins vary in their molecular weight. For example, MCM2 is detected at approximately 125 kDa, while MCM3, MCM4, MCM5, MCM6, and MCM7 are detected at approximately 102 kDa, 97 kDa, 92 kDa, 105 kDa, and 85 kDa, respectively, by western blot analysis.

Primary Antibody

Name: MCM Antibody

Clone: Rabbit anti-Human

Supplier: Pharmingen

Catalog Number: 559541

Dilution: 1:300 using IHC-Tek^TM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval

Device: IHC-Tek^TM Epitope Retrieval Steamer Set (Cat# IW-1102)

Buffer/pH value: IHC-Tek^TM Epitope Retrieval Solution (Cat# IW-1100)

Heat/Cool Temperature: 95-100 ºC/room temperature

Heat/Cool Time: 20 minutes/20 minutes

Detection Methods

Standard Method: ABC Method or LSAB Method

Enhanced Method: Polymeric Methods

Chromogen Substrate

Reagent: DAB

Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent: Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Nuclear

Images: Search image

Additional Information:

Tissue Type: GI Tract (normal colon, colonic adenoma, colonic adenocarcinoma)

Fixation: Formalin fixed paraffin sections

Positive Control: GI Tract (normal colon, colonic adenoma, colonic adenocarcinoma)

Negative Control: Omit primary antibody, isotype control, absorption control

Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary

References:

1. Rodins K, et al (2002) Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: relationship to tumor dormancy and potential clinical utility. Clin Cancer Res 8:1075-1081.