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1. Legal
GE and GE monogram are trademarks of General Electric Company. Amersham, Hoefer, Multiphor, PlusOne, PhastGel and PhastSystem are trademarks of GE Healthcare companies.Coomassie is a trademark of Imperial Chemical Industries Ltd© 2006 General Electric Company – All rights reserved. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specification and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions
2. Handling
2.1. Safety warnings
and precautions
Warning: For research use only.Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.All chemicals should be considered as potentially hazardous. We therefore recommend that this product is
handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing, such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes, wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice
2.2. Storage
The kit should be stored at 2–8℃.
2.3. Expiry
For expiry details see outer packaging.
3. Components
Protein mixture 250 μg/vial, 10 vials contains the following proteins:Thyroglobulin (1), porcine thyroid, 76 μg, molecular weight (Mr) 669 000 Ferritin (2), equine spleen, 50 μg, Mr 440 000 Catalase (3), bovine liver, 36 μg, Mr 232 000 Lactate dehydrogenase (4), bovine heart,
48 μg, Mr 140 000. Albumin (5), bovine serum, 40 μg, Mr 66 000.
The amount of each protein has been chosen to give bands of equal intensity when stained with Coomassie? Brilliant Blue following electrophoresis. Intensities may vary when using other staining methods.
4. Other materials required
? Electrophoresis reagents appropriate to the application being run.
? Detection reagents appropriate to the application being run.
? Gel electrophoresis equipment.
5. Critical parameters
? Reconstitute the HMW standard vial in appropriate buffer.
? Not recommended for use in denaturing systems i.e. containing sodium dodecyl sulphate.
6. Description
The High Molecular Weight Calibration Kit is a lyophilized mixture of five highly purified well-characterized proteins for use in molecular weight estimation under non-denaturing conditions.
The molecular mass of the protein under investigation is determined by comparing its electrophoretic mobility with that of proteins contained in the kit.
Ten vials are supplied, each containing a lyophilized mixture of highly purified protein standards of molecular mass range (Mr) 66 000 to 669 000
7. Protocol
7.1. Preparation of calibration kit
Reconstitute the contents of a vial in 100 μl of the electrophoresis sample buffer appropriate to the application being run. When reconstituted in this volume, the protein solution will also contain about 25% sucrose. It is therefore not necessary to add sucrose, glycerol or other density enhancing agents to the sample buffer.For Coomassie Brilliant Blue detectionLoad reconstituted standards without further dilution.For silver stain detectionDilute the reconstituted proteins by at least 50-fold in the electrophoresis sample buffer appropriate to the application being run.
7.2. Gel loading
Select the appropriate sample volume from the table:Gel type Sample volume (μl)Vertical mini 5–10Vertical standard 10–20Multiphor?II flatbed 5–20，PhastSystem? 0.3–4
7.3. Electrophoresis
Perform electrophoresis according to the instructions supplied with the gel apparatus being used.
7.4. Detection
Stain the gel using the desired method.
7.5. Molecular weight determination
The electrophoretic mobility of non-denatured proteins depend on their charge and shape as well as their molecular mass. It is therefore generally not possible to estimate the molecular mass of a non-denaturing protein using the HMW Calibration Kit on a single gel. The molecular mass of a non-denaturing protein can be estimated by running multiple gels of different polyacrylamide concentrations and plotting Rf vs. acrylamide concentration for each standard (Ferguson plots (6,7)).
Note: These standards are not recommended for use in denaturing gel electrophoresis in systems containing sodium dodecyl sulphate (SDS, Laemmli gels). Some of the standards consist of multiple subunits and will dissociate under denaturing
conditions. Dissociation may not be complete. further complicating interpretation.
