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zhizhi11656木虫 (正式写手)
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[求助]
求试剂盒说明书 已有1人参与
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| 求试剂盒说明书ProteoMiner Protein Enrichment Large-Capacity Kit（BIO-RAD） 编号：1633007，非常感谢！ |
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zhizhi11656
木虫 (正式写手)
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4楼2014-06-08 11:12:43
【答案】应助回帖
★ ★ ★ ★ ★
感谢参与，应助指数 +1
zhizhi11656: 金币+5, ★★★很有帮助 2014-06-08 11:12:17
感谢参与，应助指数 +1
zhizhi11656: 金币+5, ★★★很有帮助 2014-06-08 11:12:17
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在Bio-Rad官网上面找的，不只是ProteoMiner Protein Enrichment Large-Capacity Kit的操作说明，而是整个ProteoMiner™ Protein Enrichment Kits的Instruction Manual，楼主看看有没有用。 Instructions for Use With ProteoMiner™ Large-Capacity Kits (Catalog #s 163-3007 and 163-3009) This protocol has been optimized for plasma and serum samples with protein concentrations of > 50 mg/ml (requires total protein load > 50 mg). For other sample types, please refer to Section 5: Sample Considerations. Note: A ProteoMiner sequential elution kit (catalog #163-3011) is available for researchers using SELDI or other downstream protein separation analysis methods, other than 2-D gel electrophoresis, and who wish to access additional proteins. If using the ProteoMiner sequential elution kit, refer to page 9 . Step 1 – Column Preparation Vacuum (at 16 mm Hg) can replace centrifugation for column preparation, sample binding and sample wash steps if desired. (Vacuum manifold is available through Bio-Rad, catalog #732-6470.) 1. First remove the top cap and then snap off the bottom cap from each of the spin columns you will be using. Note: Do not discard top or bottom caps, they will be reused throughout the protocol. If beads settle in top cap, replace after removing bottom plug and centrifuge with top cap on column. To use bottom cap as a plug, invert and firmly place in bottom of spin column. 2. Place the column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec to remove the storage solution. Discard collected material. Note: Kit contains one capless collection tube per spin column for the following steps: column preparation, sample binding, and sample wash. Kit contains one capped collection tube per spin column to be used for the elution step, allowing for easy storage of your eluted sample. 3. Replace the bottom cap and add 600 μl wash buffer, then replace top cap. 4. Rotate column end-to-end several times over a 5 min period. 5. Remove bottom cap, place the column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec to remove buffer. Discard collected material. 6. Repeat steps 3 and 4. 7. Remove caps, place the column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec to remove the wash buffer. Discard collected material. 8. Replace bottom cap on spin column. The column now contains 100 μl of settled beads and is ready for sample binding. Step 2 – Sample Binding Samples should be free of precipitate. If needed, centrifuge samples at 10,000 x g for 10 min to clarify. Take precautions to avoid the bottom aggregate proteins and top lipid layer when recovering your sample. It is recommended that at least 1 ml of sample (protein concentration ≥50 mg/ml) is added to the column, as lower volumes may not achieve optimal results. For other sample types, please refer to Section 5: Sample Considerations. 1. Add 1 ml of sample to column. Replace top cap and rotate column on a platform or rotational shaker for 2 hr at room temperature. Note: If using plasma, clumping may occur after 1 hr of binding; this is expected and will not negatively impact your sample preparation. Heparinized plasma is not compatible with this kit. Step 3 – Sample Wash 1. Remove bottom cap, place column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec. Discard collected material. 2. Replace the bottom cap and add 600 μl of wash buffer to column. Replace top cap and rotate from end-to-end several times over a 5 min period. 3. Remove bottom cap, place column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec. Discard collected material. 4. Repeat steps 2 and 3 three more times. Step 4 – Elution 1. After all wash buffer has been removed, replace the bottom cap and add 600 μl deionized water. 2. Attach top cap and rotate end-to-end for 1 min. 3. Remove caps, place column in a capless collection tube and centrifuge at 1,000 x g for 30–60 sec to remove water. Discard collected material. If using vacuum up to this point, you will now need to switch to centrifugation . 4. Attach bottom cap to the column (take caution to ensure the bottom cap is tightly attached). Add 100 μl of rehydrated elution reagent (refer to Section 4 for rehydration instructions) to the column and replace top cap. Lightly vortex for 5 sec. Note: For 2-D users who plan to use DIGE, this elution reagent will require clean up and pH adjustment (described in Section 11). As an alternative you may elute with DIGE buffer. However, this may result in a decreased yield and number of protein spots. 5. Incubate column at room temperature, lightly vortex several times over a period of 15 min. 6. Remove caps, place in a clean collection tube labeled E1 and centrifuge at 1,000 x g for 30–60 sec. This elution contains your eluted proteins; do not discard. 7. Repeat steps 5–6 two more times. (Elutions may be pooled or analyzed individually. If analyzed individually, you will need additional collection tubes not provided with kit.) 8. Store elution at –20°C or proceed with downstream analysis. (For best results, we recommend a clean up of your sample prior to analysis. See Section 11 for more information on preparing sample for analysis.) |
2楼2014-06-08 00:32:34
3楼2014-06-08 00:33:51
zhizhi11656
木虫 (正式写手)
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5楼2014-06-08 15:29:54
