发布时间 : 星期一 文章厦门大学生命科学学院-厦门大学生物医学仪器共享平台更新完毕开始阅读
所有样品必须注明样品中的蛋白量（多少mg，ug或者ng）
附Commassie法和BCA法蛋白定量方法说明 Commassie法优点是：
1.灵敏度高，据估计比Lowry法约高四倍，其最低蛋白质检测量可达1ug。这是因为蛋白质与染料结合后产生的颜色变化很大，蛋白质－染料复合物有更高的消光系数，因而光吸收值随蛋白质浓度的变化比Lowry法要大的多。
2.测定快速、简便，只需加一种试剂。完成一个样品的测定，只需要5分钟左右。由于染料与蛋白质结合的过程，大约只要2分钟即可完成，其颜色可以在1小时内保持稳定，且在5分钟至20分钟之间，颜色的稳定性最好。
3.干扰物质少。如干扰Lowry法的K+、Na+、Mg2+离子、Tris缓冲液、糖和蔗糖、甘油、巯基乙醇、EDTA等均不干扰此测定法。
此法的缺点是：
1.由于各种蛋白质中的精氨酸和芳香族氨基酸的含量不同，因此Bradford法用于不同蛋白质测定时有较大的偏差，在制作标准曲线时通常选用g—球蛋白为标准蛋白质，以减少这方面的偏差。
2. 仍有一些物质干扰此法的测定，主要的干扰物质有：去污剂、Triton X-100、十二烷基硫酸钠（SDS）和0.1M的NaOH。（如同0.1M的酸干扰Lowary法一样）。
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厦门大学生物医学仪器共享平台
网址：http://121.192.179.197 电邮：bio-center@xmu.edu.cn 地址：福建省厦门市厦门大学翔安校区黄朝阳楼D102 电话/传真：0592-2186630 邮编：361002
3. 标准曲线也有轻微的非线性，在0-1000ug/ml浓度范围内有较好线性。因而不能用Beer定律进行计算，而只能用标准曲线来测定未知蛋白质的浓度。
BCA法特点： 1. 灵敏度高，检测浓度下限达到25ug/ml，最小检测蛋白量达到0.5ug，待测样品体积为1-20uL。 2. 测定蛋白浓度不受绝大部分样品中的去污剂等化学物质的影响，可以兼容样品中高达5%的SDS，5%的Triton X-100，5%的Tween 20，60，80。 3. 在20-2000ug/ml浓度范围内有良好的线性关系。 4. 检测不同蛋白质分子的变异系数远小于考马斯亮蓝法蛋白定量。 5. 受螯合剂和略高浓度的还原剂的影响：EDTA小于10mM。DTT小于1mM巯基乙醇低于1mM
General IP protocol（from Dr. Chen Lab）
Setting up complex IP reaction for mass spectrum analysis
1. Wash anti-flag beads with 1 mL Buffer D0.3 for two times.
2. Transfer fractionated cell lysate to the 1.5ml tube containing 10-30 uL packed anti-Flag beads and incubate on rotator at 4 C for 2 h.
3. Centrifuge at 6000 rpm (4000xg ) at 4 C for 30 sec .
4. Wash beads 4 times with 1.2mL Buffer CF0.3 (IP buffer, 0.3M NaCl) slowly and wash once with Buffer D0.3 (Ka buffer). Then wash with D0.3 final buffer twice to remove detergent.
5. Remove most liquid. use a 25G5/8 needle FIRST poke a small hole in the lid of the tube to release the pressure inside and THEN a very small hole through the pointed end at the bottom of the tube. To make sure that the hole is just big enough to allow the liquid but not any beads and spin out in Eppendorf 5417C centrifuge at 6000 rpm (4000xg ) at 4 C for 0.5min.
6. The proteins on resin are denatured in equal volumn of UA elution buffer (8-15ul ), tap to mix and stand at Room Temp (RT) for 15 min with tapping several times.
7. Put tube on top of another 1.5 mL microtube. Spin out supernatant into bottom tube at 6000 rpm (4000xg ) at 4 C for 40 sec.
8. Add another 10μL UA elution buffer, mix and stand at RT for another 10-15min, spin out supernatant into the same bottom tube at 6000 rpm (4000xg ) at 4 C for 1 min.
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厦门大学生物医学仪器共享平台
网址：http://121.192.179.197 电邮：bio-center@xmu.edu.cn 地址：福建省厦门市厦门大学翔安校区黄朝阳楼D102 电话/传真：0592-2186630 邮编：361002
Buffer D0.3: 0.3M KCl/ 20mM Hepes-KOH (pH 7.9)/15% (v/v) Glycerol/ 0.2mM EDTA/ 0.2% NP-40/0.5mM PMSF*/1mM DTT*/ (*: add freshly) eliminate nonspecific binding.
D0.3 final buffer: 0.3M KCl/ 20mM Hepes-KOH (pH 7.9)/15% (v/v) Glycerol/ 0.2mM EDTA/0.5mM PMSF*/1mM DTT*/ (*: add freshly) eliminate detergent.
UA elution buffer : 8M Urea/50mM NH4HCO3. Denature, Elution
Diversity of cell fractionations and beads washing
Buffer CF is used for cell lysing and sequential extraction. We adjust NaCl concentration to make various buffer CF, ( Buffer CF 0; CF 0.075; CF 0.1; CF 0.12; CF 0.15; CF 0.2; CF 0.25; CF 0.3; CF 0.4. ). We sequentially extract cells with various buffers (NaCl concentration from low to high) to purify target protein participated in different complex. (maybe small complex to big complex or free complex to anchored complex. ). Before IP we use 5 M NaCl to up regulate Na+ concentration to 0.3 M (or 0.4 M sometimes for strong interactions) .
Buffer D is used for reducing nonspecific binding like (beta-actin, vimentin, GAPDH…). KCl concentration must be lower than (or equial to) NaCl concentration that used in binding process. If KCl goes higher, flag-tag protein would be dissociated from flag beads and complex will disassemble.
MODIFIED SILVER STAIN & BLEACH:
For proteins of molecular weights under 50 kDA, silver staining will be necessary for simple mass spectrometric ID. Proteins under study should not be chemically modified, except perhaps to reduce and alkylate cysteine residues, which may be helpful in some cases for the digest to proceed optimally. This modified silver stain minimizes the Schiff base formation effects of aldehydes, which interfere with tryptic digestion at the C terminus of Lys and Arg and accurate mass measurement. Bleaching partially remedies the adverse effect on
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厦门大学生物医学仪器共享平台
网址：http://121.192.179.197 电邮：bio-center@xmu.edu.cn 地址：福建省厦门市厦门大学翔安校区黄朝阳楼D102 电话/传真：0592-2186630 邮编：361002
overall peptide recovery after digestion which progressively worsens with the duration of exposure.
1. Fix gel in 40% ethanol/10% acetic acid in H2O (v/v), overnight. 2. Wash for 10 min in 50% methanol (v/v).
3. Wash with water for 10 min to remove residual acid.
4. Sensitize gel by a 1-min incubation in 0.02% (w/v) sodium thiosulfate. 5. Rinse w/ 2 changes of Milli-Q H2O for 1 min each.
6. Submerge gel and incubate in 0.1% (w/v) silver nitrate for 20 min at 4?C (e.g., cold room).
7. Discard silver nitrate and rinse gel 2x w/ Milli-Q H2O for 1 min each. 8. Develop gel in 0.04% formaldehyde [dilute 108 uL of 37% formaldehyde in 100 mL of 2% (m/v) sodium carbonate)] under vigorous shaking. (If developer turns yellow, immediately discard and replace w/ fresh solution.)
9. Terminate the development by discarding the reagent and storing the gel in 1% acetic acid at 4?C until further analysis.
10. Once bands of interest have been excised from the gel, prepare destain
(“bleach”) solutions (A and B) fresh each time as follows:
a. 30 mM Potassium ferricyanide [K3Fe(CN)6]: Weight out 0.05 g and
dissolve in 5 mL Milli-Q H2O.
b. 100 mM Sodium thiosulfate pentahydrate (Na2S2O3 ?5H2O): Weight
out 0.124 g and dissolve in 5 mL Milli-Q H2O.
11. Mix equal (500-uL) volumes of solutions A and B, cover each gel piece w/
destain solution. Vortex lightly until stain disappears. 12. Rinse approx. 5x w/ H2O.
13. Cover each gel piece w/ 0.2 M NH4HCO3 in 50% acetonitrile (v/v). 14. Incubate at 37?C for 15 min. 15. Rinse approx. 5x w/ H2O.
16. Cut (dice) gel band and prepare as usual for digest.
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厦门大学生物医学仪器共享平台
网址：http://121.192.179.197 电邮：bio-center@xmu.edu.cn 地址：福建省厦门市厦门大学翔安校区黄朝阳楼D102 电话/传真：0592-2186630 邮编：361002