ReadyBlot Digestive Tract Protein explorer

Instruction Manual No. RDWB-51, ReadyBlot Digestive Tract Protein Explorer

Adult Mouse Digestive Tract; Cat. No.MDWB-41 (Information given here will also apply to rats from other age groups or similar blot from mouse). Study distribution of proteins in 13 anatomically and functionally defined regions of digestive tract with premade protein blots.

The digestive system consists of digestive tract and its associated glands. The gastrointestinal tract can be divided into the following major regions: Esophagus; Stomach (Cardia, Fundus, Body and Pylorus); Small Intestine (Duodenum, Jejunum and Ileum); Large Intestine (Cecum, Colon, Rectum, and Appendix). The entire tract is primarily composed of a lumen, which is surrounded by a wall made up of 4 principal layers: mucosa (mucous membrane), submucosa, muscularis externa and serosa. Each layer contains glands, blood and lymph vessels and lymphoid tissues The mucosa is composed of epithelial lining, lamina propria (a loose connective tissue) and two layers of smooth muscle cells (muscularis mucosae). The submucosa is composed of loose connective tissue with many submucosal nerve plexus. The muscularis externa contains 2 layers of smooth muscle cells with myenteric nerve plexus, sandwiched in-between. The serosa is composed of one inner layer of a loose connective tissue rich in adipose tissue and an outer squamous epithelial layer of covering (mesothelium).

The Esophagus, is a muscular tube through which, food-stuff is transported from mouth to stomach. It is covered by nonkeratinized, stratified squamous epithelium. It contains mucus secreting esophageal and esophageal cardiac glands in the submucosa and lamina propria layers, respectively. The Cardia, is a narrow circular band at the transition between esophagus and stomach. Its lamina propria contains tubular cardiac glands. Its parietal cells secrete HCl and other secretory glands produce mucus and lysozyme. The Fundus and the Body, contain branched tubular gastric glands in lamina propria. These glands consist of undifferentiated, mucous neck, parietal (Oxyntic), chief (Zymogenic) and enteroendocrine cells. The secretions from these cells include HCl, KCl and traces of other electrolytes, pepsin, lipase and a gastric intrinsic factor (a glycoprotein which binds avidly to vitamin B12). In fundus of the stomach, 5-hydroxytryptamine (serotonin) is one of the principal secretory products. The Pylorus, contain tubular pyloric glands secreting mucus, gastrin and lysozyme. The enteroendocrine cells of pylorus secrete somatostatin, which inhibits the release of other hormones, including gastrin. The Small Intestine begins at Duodenum. The duodenum commences at the pylorus and unites with at Jejunum. Most of the lipid absorption occurs in the duodenum and upper jejunum. The lining of small intestine has a series of permanent folds (plicae circulares) consisting of mucosa and submucosa. The plicae circulares are charesteristics of jejunum but not of duodenum or ileum. The small intestine also contains intestinal glands (crypts or glands of Lieberkuhn) and Intestinal villi which are outgrowths of the mucosa. The intestinal glands consist of several types of cells including undifferentiated, goblet, Paneth, enteroendocrine, membranous epithelial (M cells) and absorptive (microvilli) cells. M-cells endocytize and transport antigens to lymphoid cells to initiate the immune response to foreign antigens. The intestinal submucosa contains numerous tubular Duodenal glands (Brunners glands), comprised of mucous type cells. These glands secrete a polypeptide, urogastrone, which inhibits gastric acid secretion and stimulates cell proliferation. The lamina propria submucosa also contain lymphoid nodules (Peyer’s patches) which are found, mostly, in the ileum. The Large Intestine extends from the end of the ileum to the anus. It differs from small intestine in its greater caliber and the presence of appendages (Appendices epiploicae). The Cecum, commencement of the large intestine, is a large blind pouch. The open end of cecum communicates directly with the Proximal Colon, which is smaller in caliber than cecum. The Distal Colon extends from the proximal colon and is a highly movable part of the colon . It differs from small intestine in its greater caliber and the presence of appendages (Appendices epiploicae). It is almost completely invested by the peritoneum. and ends into rectum.

The Rectum is continuous above with colon and ends in the anal canal. The large intestine is responsible for passive absorption of water and formation of fecal mass. It consists of a mucosal membrane with no folds except in the Rectal portion. The cells of epithelial lining have short microvilli and Liberkuhn glands consist of large number of globlet cells, absorptive cells and a small number of enteroendocrice cells. The muscularis externa is composed of longitudinal strands which congregate into three thick bands called Teniae coli.
Acquisition of animal or human digestive tract tissue is not only time-consuming and expensive, but also requires expertise and training in anatomy, cell and molecular biology. ADI has carefully dissected and processed 13 anatomically and functionally distinct areas of digestive tract for the study of proteins using Western blots. The proteins have been electrophoresed, electro-blotted, and blocked.

Each Digestive Tract ReadyBlot has the proteins from the following regions:

Lane 1: Multi-colored Mol. Wt markers (see details below).     Lane 2: Esophagus 
Lane 3: Cardia                   Lane 4: Fundus                              Lane 5: Body 
Lane 6: Pylorus                  Lane 7: Duodenum                        Lane 8: Jejunum 
Lane 9: Ileum                     Lane 10: Cecum                             Lane 11: Proximal. Colon
Lane 12: Distal Colon        Lane13: Rectum                             Lane14: Liver
Protein Load: ~25-30 ug protein; Further optimized for equal protein load (Fig 1) and with beta-actin (Fig. 2) immuno blot.



Tissue Extracts: All procedures are performed at 2-4oC. Freshly dissected tissues were washed in 10 mM Tris, pH 7.5, 2 mM EDTA, 0.32M sucrose, and proprietary cocktail of protease inhibitors. The mucosal cells were scraped, washed, homogenized, and centrifuged at 105,000g. The clear supernatant is collected. Protein concentration determined and adjusted.

SDS-Gel Electrophoresis and blotting:Digestive tract protein extracts were mixed with 2X standard Laemmeli reducing buffer, heated for 5 min at 90oC. The proteins were run on 4-20%-reducing SDS-mini gels at 200 V for approx. 50 min. Multi-colored high range mol. Wt markers (ADI Cat # HMWM-11) were loaded in on each gel: (Aprotinin, Blue, 6.5K); (Lactalbumin, purple, 14.2K); (Soybean trypsin inhibitor, green, 20.1); (Carbonic anhydrase, orange, 29K); (Ovalbumin, yellow, 45K); (bovine serum albumin, pink, 66K); (beta-glactosidase, turquoise, 116K), and (muscle myosin, blue, 205K). The proteins were transferred to nitrocellulose or PVDF membrane using mini-transblot cells. Homogeneity of protein transfer in all 14 lanes was verified using water soluble Stain-ALL dye (ADI Cat # SALL-500) for 5 min. Protein lanes were identified and marked 1-14. Membranes were washed in PBS to remove the dye. Multi-colored mol. Wt standards (Lane -1) have been marked on the blot.

Blocking: After destaining, membranes were blocked with 1:10 diluted PBS/milk-based buffer (ADI Cat# 80062) and air-dried.

Form, Storage, and Recommended Usage: Blots are provided pre-blocked and in ready-to-use forms. Store unused blots at 4oC in a sealed bag. ReadyBlot should be handled with care as blotting membrane is quiet brittle. These blots should be used within 3-4 months. It is recommended to wet the blot first in appropriate buffer before incubating with antibodies.

This blot will be most useful for proteins that are relatively abundant in a particular region of digestive tract. Very low abundant proteins that require the use of enriched cell membranes, cytosolic or nuclear fractions may be poorly represented in this blot.

Related Products:

1. Mouse monoclonal beta-actin antibody, cat # ACTB12-M; 

Western blot recycling kit (strip antibodies in ~15 min. at room temp and re-use blots; sufficient reagents to strip 20-40 mini blots), Cat # 90100

Western blot kit (contains all necessary blocking, wash, antibody dilute, ECL reagents and a specified (anti-rabbit, mouse etc antibody conjugates; sufficient for 15-30 blots), Cat # 80200

4. High range 
multi-colored mol. Wt markers as shown on the blot, Cat # HMWM-11; 250 ul (load 5-8 ul/lane).

Western Blot Recycling Kit (Reuse ReadyBlot or any other blot many times by stripping antibodies at room temp.

All Products are for in vitro research use only.