Study distribution of Proteins in Human Tissues in Hours! 

Studies on distribution of a given protein in various tissues are carried out to find its probable function. In addition, the protein levels may vary according to the age or physiological or pathological conditions. There is also evidence of selective processing of mRNA (alternative splicing, posttranslational modifications (glycosylation, phosphorylation, etc) or interaction with other modifying partners. Therefore, mRNA and protein levels may not always correlate. It is important to study the actual levels of a given protein in various tissues. However, acquisition of animals, preparation of appropriate protein extracts, and subsequent processing of protein gels is not only time-consuming and expensive, but also requires expertise and training in tissue processing and preparation of protein samples. In order to simplify and expedite this process, ADI has carefully dissected and processed several major tissues from human. Total tissue proteins have been, extracted, 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. These blots can be part of an initial feasibility study to see if there is an interesting aspect related to a protein distribution. A more detailed, controlled study can then be initiated to elaborate initial findings. The protein blots can be used in parallel to the human multiple tissue blots (mRNA).

ReadyBlot Human Tissue protein explorer has proteins from the following tissues:

Lane 1: Mol. Wt markers (see details below)  Lane 5: Kidney  Lane 9: Pancreas
Lane 2: Brain  Lane 6: Liver  Lane 10: Spleen
Lane 3: Heart  Lane 7: Lung  Lane 11: Ovary 
Lane 4: small intestine  Lane 8: Sk. Muscle  Lane 12: Testes

Tissue Processing: Tissues samples (post-mortem) were obtained after informed consent. Tissues were stored frozen at -80oC until processed. Tissues were homogenized in an isotonic extraction buffer (Tris buffer pH 7.5, containing EDTA, sucrose and proprietary additives including several protease inhibitors), centrifuged to remove debris and nuclei, and clear supernatants (extracts) collected. The tissue extracts should contain most cytoplasmic and membrane proteins. Protein was measured and equalized with respect to total proteins (Fig. 1) and beta-actin (Fig. 2) immunostaining.

SDS-Gel Electrophoresis and blotting: Tissue protein extracts were mixed with 2X standard Laemmeli reducing buffer, heated for 5 min at 90oC. Approx 10 ug total proteins were run on 4-20%-reducing SDS-mini gels at 200 V for approx. 45 min. The proteins were transferred to PVDF membranes using mini-transblot cells. Homogeneity of protein transfer in the sample was verified using water soluble dye amido-black. Tissue Protein lanes are marked 1-11. Membranes were washed in PBS to remove the dye. The position of high range mol. wt markers (A-J; 200-6 kDa) are clearly marked on each blot.

Blocking: After destaining, membranes were blocked with 1:10 diluted PBS/milk-based buffer (ADI Cat# 80062) and air-dried. It is possible to order the blots without any blocking agents.

 

  
  Fig. 1. Total protein profiles of various tissues.


Fig 2. beta-actin profile of human tissues (beta-actin in some tissues does not react with the non-muscle type actin used in this blot).

Total protein profile of adult human tissue stained with coomassie blue (Fig. 1) beta-actin antibody (Fig. 2).

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.

All Products are for in vitro research use only.