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zhizhi11656

木虫 (正式写手)

[求助] 求试剂盒说明书 已有1人参与

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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1楼 2014-06-07 16:18:13
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寂静之林

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【答案】应助回帖

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感谢参与，应助指数 +1
zhizhi11656: 金币+5, ★★★很有帮助 2014-06-08 11:12:17
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.)
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2楼2014-06-08 00:32:34
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寂静之林

银虫 (小有名气)
不好意思，本人级别低了一点，发不了附件文件，如果楼主想要pdf原文的话，我试试能不能私信发给你吧
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3楼2014-06-08 00:33:51
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zhizhi11656

木虫 (正式写手)
引用回帖:
3楼: Originally posted by 寂静之林 at 2014-06-08 00:33:51
不好意思，本人级别低了一点，发不了附件文件，如果楼主想要pdf原文的话，我试试能不能私信发给你吧

谢谢啦！
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幸运着，努力着。。。
4楼2014-06-08 11:12:43
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zhizhi11656

木虫 (正式写手)
引用回帖:
3楼: Originally posted by 寂静之林 at 2014-06-08 00:33:51
不好意思，本人级别低了一点，发不了附件文件，如果楼主想要pdf原文的话，我试试能不能私信发给你吧

如果能私信传给我最好了，不过我没传过。
赞一下 回复此楼
幸运着，努力着。。。
5楼2014-06-08 15:29:54
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寂静之林

银虫 (小有名气)
引用回帖:
5楼: Originally posted by zhizhi11656 at 2014-06-08 15:29:54
如果能私信传给我最好了，不过我没传过。...

楼主，不好意思，刚刚才学会上传附件，试剂盒说明书现在上传给你了！
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    • 2014-06-08 21:35:41, 102.52 K
6楼2014-06-08 21:36:39
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zhizhi11656

木虫 (正式写手)
非常感谢！
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幸运着，努力着。。。
7楼2014-06-09 14:08:37
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踏实做人
8楼2014-09-03 22:04:44
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