Study distribution of Proteins in Rat Brain in Hours!


ReadyBlot Brain Protein Explorer®

Brain is one of the most complex organ in higher vertebrates. It is responsible for controlling growth, physical, mental, physiological, endocrinological, and emotional activities. The brain cells are inherently complex and obviously depends upon the ability of its cells to specialize in one or more of these activities. Brain tissues have been divided into dozens of major and hundreds of sub-areas that are anatomically and functionally distinct. The brain cells achieve these specialized activities by expressing hundreds of specific house keeping proteins, enzymes, transporters, and receptors. Expression of many genes in various areas of the brain is age and development related and may undergo an irreversible change. Therefore, it is very important to study the normal and abnormal expression of various proteins in order to delineate their physiological functions.

Acquisition of animal or human brain tissue is not only time-consuming and expensive, but also requires expertise and training in brain anatomy, cell and molecular biology. ADI has carefully dissected and processed 12 anatomically and functionally distinct areas of rat brain for the study of proteins using Western blots. The brain proteins have been electrophoresed, electro-blotted, and blocked. A lane of pre-stained mol. wt markers is included in each blot to assist you in identifying the size of the proteins.

 1. Frontal Cortex  5. Olfactory bulb  9. Entorhinal Cortex
 2. Posterior Cortex  6. Striatum  10. Pons
 3. Cerebellum  7. Thalamus  11. Medulla
 4. Hippocampus  8. Midbrain  12. Spinal Cord

Animals: Normal Adult Rat (Balb/c, Male, Av. Weight ~25-35 g).

Brain Protein Extraction: Whole Brain tissue was collected after quick decapitation and washed in cold normal saline to remove any contaminating blood. Various regions of the brain, as outlined in Fig. 1, were immediately dissected and kept in cold extraction buffer [50 mM Tris, pH 6.8, 1 mM EDTA, 2% SDS, 1 ug/ml each of protease inhibitors (leupeptin, aprotinin, and pepstatin)]. Protein concentration was equalized in samples from all brain regions.

SDS-Gel Electrophoresis and blotting: Brain tissue extracts were mixed with 2X standard Laemmeli reducing buffer, heated for 5 min. at 90oC. Brain tissue proteins (~25-30 ug) were run on 4-20%-reducing SDS-mini gels at 125 V for approx. 90 min. Pre-stained mol. Wt markers (Biorad # 161-0324) were:

A. Aprotinin, Blue, 7.5 kDa
B. Lysozyme, red,18.3 kDa
C. Soybean trypsin inhibitor, Orange 32.1 kDa
D. Carbonic anhydrase, Violet, 41.7 kDa)
E. (Bovine serum albumin, green, 85 kDa)
F. beta-galactosidase, magenta, 128 kDa)

The proteins were transferred to PVDF (0.45mm) and the homogeneity of protein transfer in lanes (1-12) was verified using water soluble Stain-ALL (ADI Cat # SALL-500). Equal amount of protein in each lane was also verified using anti-beta-actin antibody as a probe (Fig. 2). Pre-stained mol. Wt standards have been marked A-G on the blot. Top (T) and bottom (B) of the gel/blot are also marked to identify the boundaries.

Proteins (Lanes 1-12) in the extracts of different regions (1-12 in Fig.1) of brain were stained with comassie (Fig.2) or probed with beta-actin antibody (Fig.3).

Blocking: After destaining, PVDF membranes were blocked with 1:10 diluted PBS/milk-based buffer (ADI Cat# 80062) and dried using proprietary techniques to minimize damage to the membrane and proteins.

Form, Storage and Usage: Blots are provided pre-blocked and in ready-to-use forms. Store unused blots at 4oC in a sealed bag. These blots should be used within 3-4 months.

Prior to its use, the ReadyBlot should be immersed in 100% methanol for 10 seconds then in water for two minutes. Finally, the wet blot should be transferred into the desired antibody solution.

Recommended Usage

These blots will be most useful for proteins that are relatively abundant in whole brain tissue. Very low abundant proteins that require the use of enriched cell membranes or nuclear fractions may be poorly represented in whole tissue blots. An attempt has been made to equalize the protein load with beta-actin. However, antibody reactivity with beta-actin in various regions may differ due to selective posttranslational modifications) or the fact that the antibody may not react with certain actin-isoforms (e.g., muscle). It is also important to realize that there is NO protein that remains the same in ALL physiological or pathological conditions. But beta-actin, tubulin, or glyceraldehydes-3-phosphate dehydrogenase has often been used as controls. Therefore, we recommend that the researchers compare the intensity of beta-signal with the intensity of the target protein in actual blot and conditions used. Beta-actin antibody is also available.

Re-use: It is possible to re-use the blot by stripping the antibodies with the Western blot recycling kit (cat # 90100) immediately after probing and recording results. The stripped and re-blocked blots can be used immediately or stored for later use. See details of stripping kit at our web site.

Other Related Products

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. rev 40323A