ReadyBlot Adult RAT Tissues Protein Explorer

Study distribution of proteins in 10 major tissues of rat with premade protein blots 

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 rat and mouse. 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.

ReadyBlot protein explorer has the proteins from the following tissues:

 

 Lane 1: mult-colored Mol. Wt markers

 Lane 5: Liver
 Lane 2: Brain  Lane 6: Lung
 Lane 3: Heart  Lane 7: Skeletal muscle
 Lane 4: Kidney  Lane 8: Pancreas
 Lane 9: Spleen  Lane 10: Testis
 Lane 11: Thymus  


Tissue Processing: Freshly dissected and washed 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 30 ug total proteins were run on 4-20%-reducing SDS-mini gels at 200 V for approx. 45 min. Pre-stained high range mol. Wt markers (ADI Cat # HMWM-11) were loaded on each gel. The position of markers from top to bottom is:

 

 Marker#  

 Protein  MarkerColor  Mol WtkDa

 A

 Aprotinin  Blue  6.5

 B

 Lactalbumin  Purple  14.2

 C

 Soybean  trypsin inhibitor  Green  20

 D

 Carbonic anhydrase  Orange  29

 E

 Ovalbumin  Yellow  45

 F

 Bovine serum albumin  Pink  66

 G

 Beta-galactosidase  Turquoise  116

 H

 Muscle myosin  Blue  205

The proteins were transferred to PVDF or nitrocellulose using mini-transblot cells. Homogeneity of protein transfer in all 12 lanes was verified using water soluble Stain-ALL (ADI Cat # SALL-500) for 5 min. Protein lanes were identified and marked 1-11. Membranes were washed in PBS to remove the dye. Multi-colored mol. Wt standards have been marked A-H 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. It is also recommended to keep the blot is PBS+azide if blots are to be re-used (stripped) to study with another antibody.

These blots will be most useful for proteins that are relatively abundant in whole tissues. 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 tissues 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.
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. It is also recommended to keep the blot is PBS+azide if blots are to be re-used (stripped) to study with another antibody.


All Products are for in vitro research use only.