2. Handling Expiry The components of these products are stable for at least 6 months when stored under the recommended conditions.

Transcription

1 GE Healthcare Albumin and IgG Removal kit Ettan sample preparation kits and reagents. An affinity resin for specific removal of albumin and IgG from human serum. A complete set of consumables required for simultaneous removal from 10 x 15 μl samples of human serum. Larger sample volumes can also be processed using this kit Code: RPN6300

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Storage Expiry 4 3. Components 5 4. Other materials required 6 5. Description 7 6. Critical parameters 9 7. Quality control Protocols Guidelines for serum sample loading with the Albumin IgG Removal kit Alumin IgG removal kit protocol Protocol for acetone precipitation Troubleshooting guide Related products 16 2

3 1. Legal GE and GE monogram are trademarks of General Electric Company. Ettan is a trademark of GE Healthcare companies. Cibacron is a trademark of Ciba-Geigy Corp 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. http// GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK 3

4 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. Warning: Contains sodium azide Some of the components contain sodium azide in dilute solution. This substance is classified as toxic when undiluted. Dispose of waste by flushing with copious amounts of water to avoid the build up of explosive metallic azides in copper and lead plumbing Storage On receipt the resin component should be stored in a refrigerator at 2 8 C. Microspin columns and tubes should be stored at room temperature Expiry The components of these products are stable for at least 6 months when stored under the recommended conditions. 4

5 3. Components 8.5 ml of a 50/50 (v/v) resin slurry 10 empty microspin columns 10 microcentrifuge tubes and lids 5

6 4. Other materials required Reagents required Acetone for concentration/desalting if required. 6

7 5. Description The Albumin and IgG removal kit, part of the Ettan sample preparation range of kits and reagents, provides a means for specific removal of albumin and IgG from human serum samples. These two major protein components of serum represent greater than 60% of the total protein content. During gel analysis of serum, the high concentration of albumin and IgG often masks the presence of other proteins with similar isoelectric point and / or molecular weight. Therefore removal of albumin and IgG from serum samples, prior to 1 dimensional (1D) or 2 dimensional electrophoresis (2D), improves the resolution of lower abundance proteins in two ways: by enabling visualization of proteins which co-migrate with albumin and IgG. by removal of a large portion of the total serum protein, which allows an increase in the protein load of the less abundant proteins. From a 15 μl aliquot of normal human serum typically, greater than 95% of albumin and 90% IgG will be removed by treatment with the Albumin IgG removal kit. The levels of albumin and IgG removed, when larger serum volumes are processed, are given under guidelines for sample loading, see page 11. The Albumin IgG removal kit uses an antibody affinity resin in a convenient format, to rapidly and selectively remove albumin and IgG from human serum. See protocol flow chart below. The high specificity of the albumin and IgG removal, ensures lower abundance proteins are retained in the sample, in contrast to the performance of traditional Cibacron Blue based methods. 7

8 CENTRIFUGE Discard upper chamber Add Add resin resin Incubate Incubate with with Transfer Transfer slurry slurry to Concentrate/desalt / mixing mixing upper to upper chamber chamber of sample as required microspin of microspin column column Figure 1. Flow chart of Albumin IgG removal protocol 8

9 6. Critical parameters Read the entire protocol thoroughly before using the kit. Ensure the resin is in suspension when removing resin aliquots. Vigorous swirling of the resin bottle, before the removal of each aliquot is recommended, to ensure the resin remains in suspension. Remove an aliquot quickly using a wide mouthed pipette tip. During serum/resin incubation ensure the resin is kept in suspension by adequate mixing on a rotary shaker. Ensure centrifugation is performed at the correct g force, and for the required time. This allows all the liquid to be eluted from the resin following sample treatment. The resin should appear dry following centrifugation. Do not over dry acetone precipitated protein pellets. 9

10 7. Quality control Every batch of Albumin IgG removal kit is functionally tested for removal of albumin and IgG, to ensure minimal batch to batch variability. 10

11 8. Protocols 8.1. Guidelines for serum sample loading with the Albumin IgG Removal Kit The values stated below are those seen when a typical human serum sample is treated using the Albumin IgG removal kit. Human serum samples contain widely varying levels of albumin and IgG and the information below should be used for guidance only. Human serum Typical level of Typical level of sample volume albumin removed IgG removed 15 μl > 95% > 90% 30 μl > 80% > 80% 45 μl > 60 70% > 70% 8.2. Albumin IgG removal kit protocol 1. Pipette 15 μl human serum into a sample tube with lid. NB Tubes to be used for sample incubation should be of adequate size to allow good mixing of the resin / serum sample volume, to ensure the resin remains in suspension during the incubation period. Disposable 15 ml centrifuge tubes are recommended. 2. Add 750 μl of the suspended slurry to the tube containing the sample. It is essential that the gel slurry is homogenous (uniform suspension) prior to pipetting. NB When dispensing the resin it may be easier to pipette if the narrow end of the tip is cut off, this would normally be performed with scissors prior to use. 3. Mix the gel/sample mixture on a rotary shaker for a minimum of 30 minutes at room temperature. Mixing speed should be sufficient to keep the gel/sample in suspension. 11

12 NB Rapid rotary shaking is required to ensure the resin remains in suspension, speeds in the region of 250 rpm are recommended. 4. Snap off the base tip of the microspin column. Place each column into a microcentrifuge tube supplied. 5. At the end of the incubation period, make sure the resin is in suspension, and carefully pipette the gel/sample mixture into the upper chamber of the microspin column which is sitting in a microcentrifuge tube. NB Ensure all liquid is removed from the tube. If drops of liquid are splashed around the incubation tube, briefly centrifuge the incubation tube and contents (1000 rpm, 2 minutes) to collect the resin and liquid, prior to transfer to the microspin column. 6. Centrifuge at approximately 6500 g for 5 minutes. NB The resin should appear dry and powdery following centrifugation. 7. Discard the upper chamber containing the gel, and collect the filtrate. NB The approximate volume of the filtrate will be 500 μl. 8. The sample is now ready to be used immediately for further processing or stored for later use. If proteins are to be analyzed by 2 dimensional gel analysis (2D), concentration and desalting will be required. Protein samples are eluted in a dilute form, concentration/desalting may be required for samples prior to 1D gel analysis, or before use in other applications. A protocol for acetone precipitation, which concentrates and desalts the sample, is supplied on page

13 8.3. Protocol for acetone precipitation 1. Place an aliquot of acetone at -20 C at least 15 minutes prior to use. NB Approximately 2 ml of acetone is required for each sample. Ensure the tube is made of an acetone compatible material. 2. Accurately measure the volume of each sample. Divide each sample volume into two microcentrifuge tubes. Add 4 volumes of ice cold acetone to the sample volume in each tube. NB 1.5 ml hinged microcentrifuge tubes are recommended, small pellets are easily visualized using this type of tube. 3. Allow proteins to precipitate at -20 C for at least 2 hours. 4. Pellet proteins by centrifugation at approximately g ( rpm in a small bench microcentrifuge) for 10 minutes at 2 8 C. NB Place the tubes into the microcentrifuge in known orientation, place the hinged lid outwards, this assists with detection of small protein pellets. 5. Decant off the acetone, do not disturb the pellet. A clean tissue can be used to carefully remove any spots of acetone away from the pellet. 6. Allow pellets to air dry (typically 5 10 minutes at room temperature), do not over dry. NB Over dried pellets can be difficult to resuspend. 7. Resuspend precipitated samples in an appropriate buffer as required. NB Typical levels of protein recovered from a 15 μl serum sample are μg. 8. If samples are not to be used immediately, store at -20 C or -70 C until required. 13

14 9. Troubleshooting guide Problems Possible causes and solutions 1. Lower volume eluted from column than expected. 2. Lower protein recovery than expected. 1. Not all liquid has been eluted from the column. Ensure centrifugation has been performed at the correct speed. Repeat centrifugation step at a higher speed. Speeds of up to g for 10 minutes can be used. The resin was not in suspension when an aliquot was removed. A higher ratio of resin to buffer may have been removed from the stock, and therefore a lower volume of liquid was contained in the aliquot. To ensure the resin is in suspension when a sample is removed, swirl the bottle and rapidly remove a sample before the resin settles, repeat for each aliquot to be removed. Make sure the lid to the stock resin bottle is replaced when the sample is being mixed. 2. If the volume of liquid eluted from the column was also low < 480 μl), see above. The level of protein recovered following treatment will depend on the individual serum sample, as levels of albumin and IgG and total protein can vary considerably. Ensure the sample is fully eluted from the column following treatment, (see above). If acetone precipitation is performed, do not overdry pellets as this can make pellets difficult to resuspend. 14

15 Problems 3. Unable to see protein pellets following acetone precipitation. Possible causes and solutions 3. For centrifugation, place tubes in a known orientation, mark the tubes, or use tubes with hinged cap and place hinge outwards. 15

16 10. Related products Sample Grinding Kit D Quant Kit Mini Dialysis Kit 1, 250 μl Mini Dialysis Kit 1, 2 ML Mini Dialysis Kit 8, 250 μl Mini Dialysis Kit 8, 2 ML D Clean up Kit SDS Clean up Kit

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20 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway NJ USA GE Healthcare Bio-Sciences KK Sanken Bldg Hyakunincho Shinjuku-ku Tokyo Japan GE Healthcare regional office contact numbers: Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: 01 / Fax: 01 / Belgium Tel: Fax: Canada Tel: Fax: Central, East, & South East Europe Tel: Fax: Denmark Tel: Fax: Eire Tel: France Tel: Fax: Germany Tel: Fax: Italy Tel: Fax: Japan Tel: Fax: Korea Tel: Fax: Latin America Tel: Fax: Middle East & Africa Tel: Fax: Netherlands Tel: Fax: Norway Tel: Fax: Russia & other C.I.S. & N.I.S Tel: +7 (495) Fax: +7 (495) South East Asia Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerland Tel: Fax: UK Tel: Fax: USA Tel: Fax: Fax: Portugal Finland & Baltics Tel: Tel: +358-(0) Fax: Fax: +358 (0) GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK imagination at work RPN6300PL Rev B 2006