精品亚洲a∨无码专区毛片-精品亚洲aⅴ无码午夜在线-精品亚洲aⅴ无码午夜在线观看-精品亚洲aⅴ无码一区二区三区-精品亚洲aⅴ无码专区毛片-精品亚洲aⅴ在线

靶點科技(北京)有限公司

Clickchemistrytools-HPG/AHA Protein Synthesis Assay Protocol

時間:2023-10-16閱讀:660

HPG/AHA Protein Synthesis Assay Protocol Fluorescent Microscopy

Introduction

L-homopropargylglycine (HPG) and L-Azidohomoalanine (AHA) are non-radioactive alternatives to the traditional 35S-methionine which is incorporated into proteins during active protein synthesis and can be directly added to cells. Commercial HPG- and AHA-based kits used for detection of de novo synthesized proteins provide great results, but are often quite expensive and provide fixed amounts of reagents, which limits optimized or off-protocol use of these kits. Self-assembled kits are a viable alternative to commercially available kits, in particular when all of the components are widely available from a number of suppliers. The amounts of reagents and the click reaction conditions are very similar between many commercial kits, and are in line with large number of published procedures for HPG- and AHA-based detection of newly synthesized proteins. Using the provided protocols, a researcher will be able to assemble an HPG or AHA Protein Synthesis Assay that would require very little, if any, fine tuning.

These kits with improved biocompatibility and detection limits were first commercialized by Thermo Fisher Scientific and sold under Click-iT® HPG and Click-iT® AHA. Click Chemistry Tools kits take advantage of next generation, copper chelating azides. The introduction of a copper-chelating moiety at the azide reporter molecule allows for a dramatic increase of the effective Cu(I) concentration at the reaction site, enhancing the weakest link in the reaction rate acceleration, greatly increasing the sensitivity and biocompatibility of HPG- and AHA-based assays for analyzing protein synthesis in cells.

Materials Required

  • HPG (L-Homopropargylglycine)(貨號:1067-25) or AHA (L-Azidohomoalanine)

  • AZDye Picolyl Azide(貨號:1254-1等別的波長)

  • Copper (II) Sulfate pentahydrate

  • THPTA.(貨號:1010-100

  • Sodium ascorbate

  • Fixative (3.7% formaldehyde in PBS)

  • Permeabilization reagent (for example, 0.5% solution of Triton®X-100 in PBS)

  • 3% BSA in PBS (pH 7.4)

  • Hoechst 33342 (optional)

Material Preparation

HPG/AHA Stock SolutionPrepare 50 mM solution of HPG or AHA in DMSO or water, for example to make 1 mL of 50 mM stock solution dissolve 8 mg of HPG or 9 mg of AHA in 1 mL of DMSO or water
AZDye Picolyl Azide Stock SolutionPrepare 1 mM solution in DMSO or water. Example: to make 150 µL, dissolve the entire AZDye Picolyl Azide Kit Pack in 150 µL of DMSO or water
Copper Catalyst (25 mM CuSO4, 62.5 mM THPTA) solutionWeight out 312 mg of Copper (II) Sulfate Pentahydrate and 1.35 g of THPTA, add 50 mL of water, vortex to dissolve completely
Reaction Buffer50 mM Tris, 150 mM NaCl, pH 7.5. Dissolve 3.02 g of Tris, 4.4 g of NaCl in 500 mL of water, adjust pH to 7.5, sterile filter
Hoechst 3334210 mg/mL stock solution. Dissolve 1 mg of Hoechst 33342 in 100 µL of deionized water
Reducing AgentDissolve 20 mg of sodium ascorbate in 1.8 mL of deionized water. Vortex until completely dissolved. Sodium ascorbate solution is susceptible to oxidation. We recommend always using freshly prepared solution of sodium ascorbate
Wash buffer0.5 mM EDTA, 2 mM NaN3 in PBS. Add 1 mL of 0.5 M EDTA and 0.13 g of dry sodium azide to 1 L of PBS. Sterile filter for long term storage

1. Cell labeling with HPG/AHA

This protocol is based on a large number of publications of HPG- and AHA-based procedures for analyzing peptide synthesis in cells used with different types of cells. An optimized HPG/AHA concentration is 50 μM but may need adjustment depending on the given cell type. Growth medium, cell density, cell type variations, and other factors may influence labeling. Investigators are encouraged to determine the optimal concentration of the HPG reagent as well as labeling time individually for each cell type on a small-scale first.

  1. Plate the cells on coverslips at the desired density and allow them to recover overnight before additional treatment

  2. Prepare 50 mM solution of HPG or AHA in DMSO or water

  3. Wash cells once with PBS, add methionine-free medium and incubate the cells at 37°C for 30–60 minutes to deplete methionine reserves

  4. Add desired amount of HPG or AHA to cells in L-methionine-free culture medium to achieve optimal working HPG/AHA concentration (50 μM, if not optimized)

  5. During addition of HPG or AHA to cells in culture, avoid disturbing the cells in ways that may disrupt the normal cell cycling patterns

  6. Incubate the cells for the desired length of time under conditions optimal for the cell type. Different cell types may require different incubation periods for optimal labeling with HPG or AHA. As a starting point we recommend 50 μM HPG or AHA for 1 hour

  7. Proceed immediately to Cell fixation and permeabilization

2. Cell fixation and permeabilization

The following protocol is provided for the fixation step using 3.7% formaldehyde in PBS followed by a 0.5% Triton® X-100 permeabilization step. Protocols using other fixation/permeabilization reagents, such as methanol and saponin, can also be used.

  1. Transfer each coverslip into a single well. For convenient processing, use 6-well plates

  2. After metabolic labeling, remove the media and add 1 mL of 3.7% formaldehyde in PBS to each well containing the coverslips. Incubate for 15 minutes at room temperature

  3. Remove the fixative and wash the cells in each well twice with 1 mL of 3% BSA in PBS

  4. Remove the wash solution. Add 1 mL of 0.5% Triton® X-100 in PBS to each well, then incubate at room temperature for 20 minutes

3. HPG/AHA detection

Note: 500 μL of the reaction cocktail is used per coverslip. A smaller volume can be used as long as the remaining reaction components are maintained at the same ratios.

  1. Prepare the required amount of the reaction cocktail according to Table 1. Add the ingredients in the order listed in the table. Use the reaction cocktail within 15 minutes of preparation.

    Table 1

    Reaction compo-nentNumber of coverslips
    1245102550
    1x Reaction Buffer
    (Material preparation)
    430 µL860 µL1.7 mL2.2 mL4.3 mL10.7 mL21.4 mL
    Copper Catalyst
    (Material preparation)
    20 µL40 µL80 µL100 µL200 µL500 µL1 mL
    Picolyl Azide Solution
    (Material preparation)
    2.5 µL5 µL10 µL12.5 µL25 µL62.5 µL125 µL
    Reducing Agent
    (Material preparation)
    50 µL100 µL200 µL250 µL500 µL1.25 µL2.5 mL
    Total Volume500 µL1 mL2.0 mL2.5 mL5.0 mL12.5 mL25 mL


  2. Remove the permeabilization buffer (step 2.4). Wash the cells in each well twice with 1 mL of 3% BSA in PBS. Remove the wash solution.

  3. Add 0.5 mL of the Reaction Cocktail to each well containing a coverslip. Rock the plate briefly to ensure that the reaction cocktail is distributed evenly over the coverslip.

  4. Protect from light, and incubate the plate for 30 minutes at room temperature

  5. Remove the reaction cocktail. Wash each well once with 1 mL of 3% BSA in PBS. Remove the wash solution.

  6. Wash each well once with 1 mL of Wash Buffer. Remove the wash solution.

  7. Wash each well once with 1 mL of PBS. Remove the wash solution.
    At this point the samples are ready for DNA staining. If no DNA staining is desired, proceed to Imaging
    If antibody labeling of the samples is desired, proceed to labeling according to manufacturer’s recommendations. Keep the samples protected from light during incubation.

4. DNA staining

  1. Wash each well with 1 mL of PBS. Remove the wash solution.

  2. repare 1x Hoechst 33342 solution by diluting stock solution of Hoechst 33342 1:2000. The final concentration of 1x Hoechst 33342 solution is 5 µg/mL.
    Final concentrations of 1x Hoechst 33342 may range from 2 μg/mL to 10 μg/mL.

  3. Add 1 mL of 1x Hoechst 33342 solution per well. Protect from light. Incubate for 30 minutes at room temperature.

  4. Remove the Hoechst 33342 solution.

  5. Wash each well twice with 1 mL of PBS.

  6. Remove the wash solution.

Imaging

Labeled cells are compatible with all methods of slide preparation

Selected References:
  1. Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A., & Schuman, E. M., et al. (2006). Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proceedings of the National Academy of Sciences of the United States of America, 103 (25), 9482-87. 

會員登錄

X

請輸入賬號

請輸入密碼

=

請輸驗證碼

收藏該商鋪

X
該信息已收藏!
標簽:
保存成功

(空格分隔,最多3個,單個標簽最多10個字符)

常用:

提示

X
您的留言已提交成功!我們將在第一時間回復您~

以上信息由企業自行提供,信息內容的真實性、準確性和合法性由相關企業負責,化工儀器網對此不承擔任何保證責任。

溫馨提示:為規避購買風險,建議您在購買產品前務必確認供應商資質及產品質量。

撥打電話
在線留言
主站蜘蛛池模板: 亚洲精品乱码久久久久久自慰| 欧美性久久| 精品网站一区二区三区网站| 国产精品久久久天天影视香蕉 | 国产一区免费在线观看| 国产乱码卡二卡三卡4| 无人区码一码二码三码在线| 欧美老妇性生活| 国产精品国产三级| 日本午夜成年在线网站| 四虎永久在线精品免费观看| 成人性生交大片免费看vr| 欧美日韩免费一区二区三区播放| 美女裸乳裸体无遮挡免费A片软件 美女裸身大乳图片大全 | 风流少妇与黑人做爰| 人妻av久久一区波多野结衣| 日本无码人妻精品一区二区视频| 国产91精品一区二区| 区产品乱码芒果精品综合| 国产玩弄放荡人妇系列| 成年女人视频在线| 女人18毛多水多A片视频| 国产精品亚洲精品久久品| GAY亚洲男男GV在线观看网站 | 给啪啪视频免费观看| av免费在线观看网址入口| 久久亚洲国产高清| 国精品人妻无码一区二区三区喝尿| 欧洲国产成人精品91软件| 精品欧美成人高清在线观看| 亚洲狼人伊人中文字幕| 国产成人精品视频午夜| 国产精品久久久久久人| 人人爱夜夜爽日日做视频| 久久草这在线观看免费| 波多野吉衣亚洲| 老湿机免费体验| 亚洲永久免费精品网站一亚| 插日本女人| 国产人妻人伦精品9| 天美传媒影视|