搜索

Polysome Profiling Analysis
多核糖体图谱分析   

评审
匿名评审
下载 PDF 引用 收藏 2 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.

Keywords: Translation(翻译), Polysome(多核糖体), MRNA(mRNA), Ribosome(核糖体), Genome-wide analysis(全基因组分析)

Materials and Reagents

  1. Sucrose
  2. 1 M HEPES-KOH (pH 7.6)
  3. 2 M KCl
  4. 1 M MgCl2
  5. PBS
  6. 1 M Tris-HCl (pH 7.5)
  7. 10 mg/ml cycloheximide (Sigma-Aldrich, catalog number: C7698 ) in MilliQ water
    Note: Cycloheximide inhibits protein synthesis by blocking translation elongation. This molecule interferes with the translocation of tRNAs with the mRNA and the ribosome, resulting in fixed ribosomes on mRNAs.
  8. Protease inhibitor cocktail (EDTA-free) (F. Hoffmann-La Roche, catalog number: 04 693 159 001 )
  9. RNase inhibitor (RNasin 40 units/μl) (Promega Corporation, catalog number: N2511 )
  10. 10% Triton X-100
  11. 10% Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  12. RNaseZap (Life Technologies, Ambion®, catalog number: AM9780 )
  13. Trizol (Life Technologies, InvitrogenTM)
  14. Oligo dT primer and Super Script III (Life Technologies, InvitrogenTM, catalog number: 18418012 and 18080093 )
  15. 10x sucrose gradient buffer (see Recipes)
  16. 10-50% sucrose solutions (see Recipes)
  17. Hypotonic buffer (see Recipes)

Equipment

  1. Ultra centrifuge and rotor (Beckman, model: Optima L-80 ; SW40Ti rotor )
  2. UV detector and fraction collector (Teledyne ISCO)
  3. NanoDrop (Thermo Fisher Scientific)
  4. 0.22 µm filter

Software

  1. TracerDAQ software (MicroDAQ)

Procedure

I.   Preparation of sucrose gradients

  1. Prepare 50 ml of 60% (w/v) sucrose solution in MilliQ water and 10 ml of 10x sucrose gradient buffer. Filter solutions with 0.22 μm filter.
  2. Prepare 10-50% sucrose solutions using 60% sucrose. Prepare gradients at least one day before cell lysis to allow gradient to diffuse overnight at 4 °C.
  3. Carefully add 1.2 ml of each sucrose solution (starting with the 50% sucrose solution) to Beckman tubes (12 ml total volume).To improve efficacy at this step, the gradients can be flash frozen in liquid nitrogen following each addition of sucrose solution. This helps in generating equally poured gradients and prevents too much diffusion.
  4. Cover tube with parafilm and incubate at 4 °C overnight. Alternatively, sucrose gradients can be stored at -80 °C indefinitely.


II.  Isolation of polysomes

  1. Prepare cells at least one day before lysing. Confluency is very important. About 80-90% confluency gets the maximum polysomes. 1-5 15-cm dishes are needed to see polysome profile.
  2. Prior to lysing the cells, treat cells with 100 μg/ml cycloheximide at a final concentration for 5 min.
  3. Wash cells twice with 10 ml of ice-cold 1x PBS containing 100 μg/ml cycloheximide.
  4. Scrape cells with 5 ml of ice-cold 1x PBS containing 100 μg/ml cycloheximide, collect them in 15 ml tube and rinse with 5 ml of ice-cold 1x PBS containing 100 μg/ml cycloheximide.
  5. Centrifuge cells at 1,200 rpm (300 x g) for 5 min at 4 °C.
  6. Aspirate supernatant, resuspend cells in 425 μl of hypotonic buffer and transfer all to a new pre-chilled 1.5 ml tube.
  7. Then add:
    5 μl of 10 mg/ml cycloheximide
    1 μl of 1 M DTT
    100 units of RNasin
  8. Vortex for 5 sec.
  9. Then add:
    25 μl of 10% Triton X-100
    25 μl of 10% Sodium Deoxycholate
  10. Vortex for 5 sec.
  11. Centrifuge at 15,000 rpm (21,000 x g, a maximal speed on a bench top centrifuge in 1.5 ml eppendorf tube) for 5 min at 4 °C.
  12. Transfer supernatant (about 500 μl) to a new pre-chilled 1.5 ml tube. Measure OD260nm for each sample using NanoDrop.


    Figure 1. Polysome profiling from MEFs treated with DMSO or Ink1341 (250 nM) for 8 h from Dowling et al. (2010). 40 S, 60 S and 80 S denote the corresponding ribosomal subunits and monosome, respectively.

  13. Load the same OD amount of lysate onto each gradient (10-30 OD260nm is loaded). Keep 10% of lysates as an input.
  14. Weight and balance each gradient.
  15. Centrifuge at 35,000 rpm for 2 h at 4 °C using SW40Ti rotor in a Beckman Coulter (Optima L-80 ultra centrifuge). No brake of brake at 1 (maximum) for deceleration. Acceleration does not matter much.
  16. While the samples are centrifuging, clean fraction collector with warm MilliQ water containing a bit of RNase ZAP (a few sprays).
  17. Carefully remove tubes from the rotor and place them at 4 °C until they are ready for running.
  18. Switch on computer, pump, UV detector and fraction collector. Set pump at 1.5 ml/min and the fraction collector by time. Place 2 ml tubes on fraction collector.
  19. Open TracerDAQ program.
  20. Run chasing solution (60% (w/v) sucrose with bromophenol blue) through the system until it reaches the needle. Make sure to see at least one drop coming out of the needle such that no bubbles are introduced into the gradient.
  21. Screw each tube onto the ISCO UV detector and then pierce the tube with the needle.
  22. Begin running the chasing solution through the gradient. Run solution with the pump at 1.5 ml/min. Click on acquire data button and press run on the fraction collector.
  23. Place fractions on dry ice or liquid nitrogen.
  24. Add 750 μl of Trizol for RNA isolation.


III. RNA isolation and RT-qPCRMQ

  1. Add 150 μl of chloroform to each fraction.
  2. Isolate RNA according to manufacturer’s Trizol protocol.
  3. Measure the RNA concentration of each fraction or input.
  4. Prepare the following solutions in 500 μl tubes or PCR tubes.
    0.5 μg of RNA                     x μl
    Oligo (dT) primer                1 μl
    dNTP (10 mM each)           1 μl
    Water x μl/total                   13 μl
  5. 65 °C for 5 min.
  6. On ice for at least 1 min.
  7. Add the following solutions (7 μl premix) on ice. Make premix before adding.
    5x buffer                    4 μl
    1 M DTT                    1 μl
    RNasin 40 units/μl     1 μl
    RTase                       1 μl
  8. Mix gently and spindown on ice.
  9. 50 °C for 60 min.
  10. 70 °C for 15 min.
  11. Keep at 4 °C until next step.
  12. Add 180 μl of MQ.
  13. qPCR according to manufacturer’s protocol.
    2x SYBR                  10 μl
    5’ Primer (10 μM)     0.4 μl
    3’ Primer (10 μM)     0.4 μl
    RT sample               5 μl
    MQ 4.2 μl/Total        20 μl

Recipes

  1. 10x sucrose gradient buffer (50 ml)
    200 mM HEPES (pH 7.6)
    1 M KCl
    50 mM MgCl2
    100 μg/ml cycloheximide
    1x protease inhibitor cocktail (EDTA-free)
    100 units/ml RNase inhibitor
  2. 10-50% sucrose solutions
    Final (%)
    60% stock (ml)
    water (ml)
    10x sucrose gradient buffer (ml)
    50
    8.3
    0.7
    1
    45
    7.5 1.5
    1
    40
    6.7
    2.3
    1
    35
    5.8
    3.2
    1

    30
    5.0
    4.0
    1

    25
    4.2
    4.8
    1

    20
    3.3
    5.7
    1

    15
    2.5
    6.5
    1

    10
    1.7
    7.3
    1

  3. Hypotonic buffer
    5 mM Tris-HCl (pH 7.5)
    2.5 mM MgCl2
    1.5 mM KCl
    1x protease inhibitor cocktail (EDTA-free)

References

  1. Alain, T., Morita, M., Fonseca, B. D., Yanagiya, A., Siddiqui, N., Bhat, M., Zammit, D., Marcus, V., Metrakos, P., Voyer, L. A., Gandin, V., Liu, Y., Topisirovic, I. and Sonenberg, N. (2012). eIF4E/4E-BP ratio predicts the efficacy of mTOR targeted therapies. Cancer Res 72(24): 6468-6476. 
  2. Dowling, R. J., Topisirovic, I., Alain, T., Bidinosti, M., Fonseca, B. D., Petroulakis, E., Wang, X., Larsson, O., Selvaraj, A., Liu, Y., Kozma, S. C., Thomas, G. and Sonenberg, N. (2010). mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328(5982): 1172-1176.
  3. Larsson, O., Morita, M., Topisirovic, I., Alain, T., Blouin, M. J., Pollak, M. and Sonenberg, N. (2012). Distinct perturbation of the translatome by the antidiabetic drug metformin. Proc Natl Acad Sci U S A 109(23): 8977-8982. 

简介

多聚体概况分析是允许监测mRNA在细胞和组织中的翻译活性的方法。 一旦收集每个多核糖体级分,使用各种分子生物学技术如Northern印迹,RT-PCR,微阵列或深度测序分析每个mRNA的翻译活性。

关键字:翻译, 多核糖体, mRNA, 核糖体, 全基因组分析

材料和试剂

  1. 蔗糖
  2. 1 M HEPES-KOH(pH 7.6)
  3. 2 M KCl
  4. 1 M MgCl 2
  5. PBS
  6. 1 M Tris-HCl(pH 7.5)
  7. 10mg/ml放线菌酮(Sigma-Aldrich,目录号:C7698)在MilliQ水中的溶液 注意:环己酰亚胺通过阻断翻译延伸来抑制蛋白质合成。 该分子干扰tRNA与mRNA和核糖体的易位,导致mRNA上的固定核糖体。
  8. 蛋白酶抑制剂混合物(无EDTA)(F.Hoffmann-La Roche,目录号:04 693 159 001)
  9. RNA酶抑制剂(RNasin 40单位/μl)(Promega Corporation ,目录号:N2511)
  10. 10%Triton X-100
  11. 10%脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
  12. RNaseZap(Life Technologies,Ambion ,目录号:AM9780)
  13. Trizol(Life Technologies,Invitrogen TM
  14. Oligo dT引物和Super Script III(Life Technologies,Invitrogen TM ,目录号:18418012和18080093)
  15. 10x蔗糖梯度缓冲液(见配方)
  16. 10-50%蔗糖溶液(见配方)
  17. 低密度缓冲区(参见配方)

设备

  1. 超离心机和转子(Beckman,型号:Optima L-80; SW40Ti转子)
  2. UV检测器和馏分收集器(Teledyne ISCO)
  3. NanoDrop(赛默飞世尔科技)
  4. 0.22μm过滤器

软件

  1. TracerDAQ软件(MicroDAQ)

程序

I.   蔗糖梯度的制备

  1. 准备50ml的60%(w/v)蔗糖的MilliQ水溶液和10ml的10x蔗糖梯度缓冲液。 用0.22μm过滤器过滤溶液。
  2. 使用60%蔗糖制备10-50%蔗糖溶液。 在细胞裂解前至少一天准备梯度,使梯度在4℃扩散过夜
  3. 小心地将1.2ml的每种蔗糖溶液(以50%蔗糖溶液开始)加入到Beckman管(12ml总体积)中。为了改善该步骤的功效,每次加入蔗糖溶液后,可以在液氮中将梯度快速冷冻。 这有助于产生同样倾斜的梯度并防止过多的扩散
  4. 覆盖管用石蜡膜并在4℃孵育过夜。 或者,蔗糖梯度可以无限期地储存在-80℃


II。  多核糖体的分离

  1. 在裂解前至少一天准备细胞。 汇合是非常重要的。 约80-90%汇合获得最大多核糖体。 1-5需要15厘米的菜肴来观察多核糖体谱
  2. 在裂解细胞之前,用100μg/ml放线菌酮处理细胞,最终浓度为5分钟
  3. 用10ml含有100μg/ml放线菌酮的冰冷的1×PBS洗涤细胞两次
  4. 用5ml含有100μg/ml放线菌酮的冰冷的1×PBS刮细胞,将其收集在15ml管中,并用5ml含有100μg/ml放线菌酮的冰冷的1×PBS冲洗。
  5. 在4℃下以1,200rpm(300xg)离心细胞5分钟。
  6. 吸出上清液,重悬细胞在425微升低渗缓冲液,并将所有转移到新的预冷1.5毫升管。
  7. 然后添加:
    5μl10mg/ml放线菌酮
    1μl1 M DTT
    100单位RNasin
  8. 涡旋5秒。
  9. 然后添加:
    25μl10%Triton X-100 25μl10%脱氧胆酸钠
  10. 涡旋5秒。
  11. 在4℃下以15,000rpm(21,000×g,在台式离心机在1.5ml eppendorf管中的最大速度)离心5分钟。
  12. 转移上清(约500微升)到新的预冷1.5毫升管。 使用NanoDrop测量每个样品的OD <260nm 。


    图1.来自Dowling等人的用DMSO或Ink1341(250nM)处理8小时的MEF的多聚体概况分析。 (2010)。 40S,60S和80S分别表示相应的核糖体亚基和单体。
  13. 将相同OD量的裂解物加载到每个梯度(加载10-30 OD 260nm)。 保持10%的裂解物作为输入
  14. 每个渐变的重量和平衡。
  15. 使用SW40Ti转子在Beckman Coulter(Optima L-80超离心机)中在4℃下以35,000rpm离心2小时。 无制动器制动,在1(最大)减速。 加速不重要。
  16. 当样品离心时,用含有一点RNase ZAP(少量喷雾)的热MilliQ水清洗馏分收集器。
  17. 小心地从转子上取下管子,并将其放置在4°C,直到它们准备好运行
  18. 打开计算机,泵,UV检测器和馏分收集器。 按1.5 ml/min设置泵,按时间设置馏分收集器。 将2 ml试管置于馏分收集器上。
  19. 打开TracerDAQ程序。
  20. 运行追踪溶液(60%(w/v)蔗糖与溴酚蓝)通过系统,直到它到达针。 确保至少有一滴液体从针头中流出,以便没有气泡进入梯度
  21. 将每根管子拧到ISCO UV检测器上,然后用针刺穿管子
  22. 开始通过梯度运行追逐解决方案。 使用泵以1.5ml/min运行溶液。 点击获取数据按钮,并在馏分收集器上运行
  23. 将馏分置于干冰或液氮中
  24. 加入750μlTrizol用于RNA分离


III。 RNA分离和RT-qPCRMQ

  1. 每部分加入150μl氯仿。
  2. 根据制造商的Trizol方案分离RNA
  3. 测量每个馏分或输入的RNA浓度
  4. 在500μl管或PCR管中制备以下溶液。
    0.5μgRNA                    x微博
    Oligo(dT)引物                1微升
    dNTP(各10mM)         1微升
    水xμl/total                  13微升
  5. 65℃5分钟
  6. 在冰上至少1分钟。
  7. 在冰上加入以下溶液(7μl预混物)。 添加前先进行预混。
    5x缓冲区                   4μl
    1 M DTT                    1微升
    RNasin 40 units /μl    1微升
    RTase                       1微升
  8. 轻轻地混合并在冰上滚动。
  9. 50℃60分钟。
  10. 70℃15分钟
  11. 保持在4℃直到下一步。
  12. 加入180μlMQ。
  13. qPCR根据制造商的协议 2x SYBR                 10微升
    5'引物(10μM)   0.4μl
    3'引物(10μM)   0.4μl
    RT示例             5微升
    MQ 4.2μl/Total       20微升

食谱

  1. 10x蔗糖梯度缓冲液(50ml) 200mM HEPES(pH 7.6)
    1 M KCl
    50mM MgCl 2/v/v 100μg/ml放线菌酮
    1x蛋白酶抑制剂混合物(无EDTA)
    100单位/ml RNase抑制剂
  2. 10-50%蔗糖溶液
    最终(%)
    60%原料(ml)
    水(ml)
    10x蔗糖梯度缓冲液(ml)
    50
    8.3
    0.7
    1
    45
    7.5 1.5
    1
    40
    6.7
    2.3
    1
    35
    5.8
    3.2
    1

    30
    5.0
    4.0
    1

    25
    4.2
    4.8
    1

    20
    3.3
    5.7
    1

    15
    2.5
    6.5
    1

    10
    1.7
    7.3
    1

  3. 低渗缓冲液
    5mM Tris-HCl(pH7.5) 2.5mM MgCl 2 v/v 1.5 mM KCl
    1x蛋白酶抑制剂混合物(无EDTA)

参考文献

  1. Alain,T.,Morita,M.,Fonseca,BD,Yanagiya,A.,Siddiqui,N.,Bhat,M.,Zammit,D.,Marcus,V.,Metrakos,P.,Voyer,LA,Gandin, V.,Liu,Y.,Topisirovic,I.and Sonenberg,N。(2012)。 eIF4E/4E-BP比例预测mTOR靶向治疗的疗效。 Cancer Res 72(24):6468-6476。 
  2. Dowling,RJ,Topisirovic,I.,Alain,T.,Bidinosti,M.,Fonseca,BD,Petroulakis,E.,Wang,X.,Larsson,O.,Selvaraj,A.,Liu,Y.,Kozma, SC,Thomas,G。和Sonenberg,N。(2010).gregator_blank" href="http://www.ncbi.nlm.nih.gov/pubmed/20508131"> mTORC1介导的细胞增殖,但不是细胞生长,由4E-BP控制。 Science 328(5982):1172-1176。
  3. Larsson,O.,Morita,M.,Topisirovic,I.,Alain,T.,Blouin,M.J.,Pollak,M。和Sonenberg,N。 抗糖尿病药物二甲双胍对视神经的明显扰动。美国国家癌症研究所Sci USA 109(23):8977-8982。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Morita, M., Alain, T., Topisirovic, I. and Sonenberg, N. (2013). Polysome Profiling Analysis. Bio-protocol 3(14): e833. DOI: 10.21769/BioProtoc.833.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

Yingying Lin
Xiamen University
Dear all,
I am a doctoral student of Xiamen University in China. Recently, our lab bought a BioComp gradient machine. And I have done several times of polysome profile of MCF-7 cell line in order to set up the technique. However, the result didn't comes out good. Here I present a graph of the latest polysome profile I did. I use the protocol you provide. I used about 370 ug of RNA to load to the 5~50% sucrose gradient and centrifuge at 36,000 rpm for 2 hr. The polysome peaks didn't drop down to the baseline at the end, and after 80S the wave didn't go down as well. What do you think the problem is? Do you have some advice for me to improve the technique? Thank you so much!


Best regards,

Ying
12/23/2015 10:03:21 PM Reply
Ivan Topisirovic
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

Looks like you are overloading the gradient and/or that there be something wrong with your gradients. What we typically load is about 10OD at 254nm. Note that we use Biocomp gradient master but we fractionate using ISCO collector and not Biocomp (so we can't advise on Biocomp), but this shouldn't be an issue. This should help, as here you have the detailed protocol (with things that may go wrong) and whole video on how to do it.

http://www.ncbi.nlm.nih.gov/pubmed/24893926

Hope this helps, and if not I'll transfer you to Morita as I haven't done one of these in a while.

12/23/2015 10:18:52 PM


Yingying Lin
Xiamen University

Dear Ivan,
Thank you for your fast reply. Actually, I have seen the jove video you mentioned. And I done the polysome profile according to it as well, because I see your protocol is similar as that. You mean I overload the RNA to the gradient, I don't understand what is 10OD at 254 mean, I have measure the RNA with nanodrop, the OD 260 is 18.198, should I dilute the sample to adjust the OD260 to be 10, and how much volume of the sample should I load to the gradient? We use the sw41i rotor. On the other hand, there was something wrong with my gradient, what do you think is wrong?
Many thanks!

Best regards,

Ying

12/23/2015 10:39:21 PM


Ivan Topisirovic
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

I have never seen a profile like yours so it's hard to say what you're doing wrong.

Yes I would load 10 OD from 260, and if you make a gradient using Biocomp gradient master you just have to make sure that it's continuous 5-50% (you can read the manual if you're confused re: zonal vs isopycnic centrifugation and it's crucial that you use rate zonal and not ispycnic caps). These are two things that I would try, and see whether they'd help.

If you followed the JOVE video and protocol it tells you exactly how much to load:

"Transfer ultracentrifuge tubes containing sucrose gradients in pre-chilled rotor buckets. Remove 500 µl from the top of sucrose gradients. Adjust lysates so that they contain the same OD (10-20 OD at 260 nm) in 500 µl of lysis buffer (described in step 2.5) and load them onto each sucrose gradient. Note: It is important to immediately load lysates on the gradients, as this will critically improve the quality of polysome preparations."

So it seems to me that you could follow it a bit more carefully, and according to my experience if you can't find this information in the protocol that's written as clearly as JOVE or the one here, then there may be a problem of how careful you are when you do the experiment. So try to be more careful.

12/23/2015 10:56:19 PM


Yingying Lin
Xiamen University

Dear Ivan,
Thank you very much! I am so embarrased to say that I saw the sentence and it mentioned 500 ul of the sample, for the engineer who set up our machine told me to add to full of the centrifuge tube, so for several times I only load 300 ul of the sample. So I see, maybe I should just remove 500 ul of the gradient and add 500 ul of the sample. Thank you again for your advice!


Best wishes,

Ying

12/23/2015 11:11:16 PM


Yingying Lin
Xiamen University

Dear Ivan,
Today, I did another polysome profile according to the protocol of jove paper, I used 10OD 260(500 uL) RNA sample to 5~50% sucrose gradient and centrifuge at 36,000 rpm for 2 hr, but the result still not good. Accidently, I cut the impurity peak, but the wave after 80S didn't go down, I can't figure out what's wrong with it, can you help me and give me some advice? Many thanks!


Best regards,

Ying

12/25/2015 2:50:01 AM


Ivan Topisirovic
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

If you followed the protocol, then then the only thing I can think of is that this could be due to the way you make the gradient or how the collector is set up, so I'd suggest you contact Biocomp. I can't help you any further as I have no idea what's going on. Never seen a profile like this. Good luck with the experiments and sorry I couldn't help.

12/25/2015 6:56:46 AM


Yingying Lin
Xiamen University

Nevermind,you've helped me a lot! Many thanks!

12/25/2015 7:02:09 AM


laura GA
isciii

Dear Ivan,
Thank you for the protocol. I have tried it with MCF7 cells (4 of 15-cm dishes). After of lysis, the OD 260 is around 2. What is the problem?. Also, I tried it with 293T cells (10 of 10-cm dishes) and the OD 260 was 1.5 . I believe the problem doesn´t the cell number because I have a lot of cells. When I run these RNA samples in a sucrose gradient I get a good profile but with low OD 254. Then, the problem is the celular lysis?
Best

3/22/2016 5:05:17 AM


Carmine Onofrillo
Bologna University
Dear all,
i'm a post doc of bologna university. Recently we bought a teledyne isco gradient system, provided with UA6 UV detector, optical unit, and a fraction collector for polysome profile analysis. In your protocols you indicate the tracerdaq as the software used for computetional data recording. I'm trying to connect UA6 detector to a personal computer to have digitalized data. I have some problems setting the tracerdaq software and the coupled hardware device, the minilab 1008. In particular i don't know how to connect output recorders of the UA6 detector to the minilab 1008, and how to set the voltage range of tracerdaq. Can you help me in some way?
Many thanks and regards.
Carmine Onofrillo.

1/23/2015 5:26:39 AM Reply
Ivan Topisirovic
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Canada

Dear Carmine,

In our lab, USB-1208L is used to connect the UA6 detector to the laboratory computer. To connect the UA6 detector to USB digital I/O, this protocol may be of use (http://www.isco.com/pcfiles/PartPDF/SL000004/UP000ZUZ.pdf). Moreover, TracerDAQ software is included in USB-1208L with the protocol about the connection (http://www.microdaq.com/measurement_computing/usb_data_acquisition/multifunction/usb-1208/usb-1208ls-daq.php). If you need any further information, please do not hesitate to contact us.

All the best,

Masahiro Morita

1/26/2015 11:54:47 AM


Carmine Onofrillo
Bologna University

Dear dott. Morita,
thanks for your fast reply. Actually i'm not sure about the connection of the cable from recorder output of UA6 he UA6 manual, detector to my device . Our device (as yours) can operate in single ended mode ( only +/- 10V) or in differential mode (+/- 10, 5, 2.5 ,2, 1.25, 1V), in single ended i have to use only 2 cable (POSITIVE and GROUND), in differential i have to connect 3 cables ( HI, LOW and GROUND). Do you know what settings i have to use? In The tracerdaq software and in instacal it is possible to check the type of connection (single ended or differential), in the settings menu. I hope you can help me.
Best regards,
Carmine Onofrillo.

1/27/2015 1:34:48 AM


Masahiro Morita
University of Texas Health Science Center at San Antonio

Dear Carmine,

I would like to send the photos of UA6 detector and USB I/O adaptor to you. Would it be possible to let me know your email address?

All the best,

Masahiro Morita

1/28/2015 7:14:29 PM