Clickchemistrytools-HPG/AHA Protein Synthesis Assay Protocol
hpg/aha protein synthesis assay protocol fluorescent microscopyintroductionl-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 requiredhpg(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 preparationhpg/aha stock solution prepare 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 solution prepare 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) solution weight out 312 mg of copper (ii) sulfate pentahydrate and 1.35 g of thpta, add 50 ml of water, vortex to dissolve completely
reaction buffer 50 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 33342 10 mg/ml stock solution. dissolve 1 mg of hoechst 33342 in 100 µl of deionized water
reducing agent dissolve 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 buffer 0.5 mm edta, 2 mm nan3in 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/ahathis 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.
plate the cells on coverslips at the desired density and allow them to recover overnight before additional treatment
prepare 50 mm solution of hpg or aha in dmso or water
wash cells once with pbs, add methionine-free medium and incubate the cells at 37°c for 30–60 minutes to deplete methionine reserves
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)
during addition of hpg or aha to cells in culture, avoid disturbing the cells in ways that may disrupt the normal cell cycling patterns
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
proceed immediately tocell fixation and permeabilization
2. cell fixation and permeabilizationthe 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.
transfer each coverslip into a single well. for convenient processing, use 6-well plates
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
remove the fixative and wash the cells in each well twice with 1 ml of 3% bsa in pbs
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 detectionnote: 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.
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 µl 860 µl 1.7 ml 2.2 ml 4.3 ml 10.7 ml 21.4 ml
copper catalyst(material preparation) 20 µl 40 µl 80 µl 100 µl 200 µl 500 µl 1 ml
picolyl azide solution(material preparation) 2.5 µl 5 µl 10 µl 12.5 µl 25 µl 62.5 µl 125 µl
reducing agent(material preparation) 50 µl 100 µl 200 µl 250 µl 500 µl 1.25 µl 2.5 ml
total volume 500 µl 1 ml 2.0 ml 2.5 ml 5.0 ml 12.5 ml 25 ml
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.
add 0.5 ml of thereaction cocktailto each well containing a coverslip. rock the plate briefly to ensure that the reaction cocktail is distributed evenly over the coverslip.
protect from light, and incubate the plate for 30 minutes at room temperature
remove the reaction cocktail. wash each well once with 1 ml of 3% bsa in pbs. remove the wash solution.
wash each well once with 1 ml ofwash buffer. remove the wash solution.
wash each well once with 1 ml of pbs. remove the wash solution.at this point the samples are ready fordna staining. if nodna stainingis desired, proceed toimagingif 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 stainingwash each well with 1 ml of pbs. remove the wash solution.
repare 1xhoechst 33342 solutionby diluting stock solution ofhoechst 333421:2000. the final concentration of 1xhoechst 33342 solutionis 5 µg/ml.final concentrations of 1xhoechst 33342may range from 2 μg/ml to 10 μg/ml.
add 1 ml of 1xhoechst 33342 solutionper well.protect from light. incubate for 30 minutes at room temperature.
remove the hoechst 33342 solution.
wash each well twice with 1 ml of pbs.
remove the wash solution.
imaginglabeled cells are compatible with all methods of slide preparation
selected references: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.
淺談自動萃取器
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直埋保溫無縫管詳細參數
二手噴霧干燥制粒機在制藥廠食品廠的應用
MT/T748-2007工業型煤冷壓強度測試方法
Clickchemistrytools-HPG/AHA Protein Synthesis Assay Protocol
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FQ641F46氣動釜底襯氟放料球閥的用途和工作原理
袋式過濾器損壞的原因
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從這個方面竟然可以了解到油顆粒度分析儀的優勢
【時訊科普】電子天平專業術語
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黑色5月！行業變局下，18家醫藥企業高管離職
materials requiredhpg(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 preparationhpg/aha stock solution prepare 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 solution prepare 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) solution weight out 312 mg of copper (ii) sulfate pentahydrate and 1.35 g of thpta, add 50 ml of water, vortex to dissolve completely
reaction buffer 50 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 33342 10 mg/ml stock solution. dissolve 1 mg of hoechst 33342 in 100 µl of deionized water
reducing agent dissolve 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 buffer 0.5 mm edta, 2 mm nan3in 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/ahathis 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.
plate the cells on coverslips at the desired density and allow them to recover overnight before additional treatment
prepare 50 mm solution of hpg or aha in dmso or water
wash cells once with pbs, add methionine-free medium and incubate the cells at 37°c for 30–60 minutes to deplete methionine reserves
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)
during addition of hpg or aha to cells in culture, avoid disturbing the cells in ways that may disrupt the normal cell cycling patterns
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
proceed immediately tocell fixation and permeabilization
2. cell fixation and permeabilizationthe 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.
transfer each coverslip into a single well. for convenient processing, use 6-well plates
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
remove the fixative and wash the cells in each well twice with 1 ml of 3% bsa in pbs
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 detectionnote: 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.
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 µl 860 µl 1.7 ml 2.2 ml 4.3 ml 10.7 ml 21.4 ml
copper catalyst(material preparation) 20 µl 40 µl 80 µl 100 µl 200 µl 500 µl 1 ml
picolyl azide solution(material preparation) 2.5 µl 5 µl 10 µl 12.5 µl 25 µl 62.5 µl 125 µl
reducing agent(material preparation) 50 µl 100 µl 200 µl 250 µl 500 µl 1.25 µl 2.5 ml
total volume 500 µl 1 ml 2.0 ml 2.5 ml 5.0 ml 12.5 ml 25 ml
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.
add 0.5 ml of thereaction cocktailto each well containing a coverslip. rock the plate briefly to ensure that the reaction cocktail is distributed evenly over the coverslip.
protect from light, and incubate the plate for 30 minutes at room temperature
remove the reaction cocktail. wash each well once with 1 ml of 3% bsa in pbs. remove the wash solution.
wash each well once with 1 ml ofwash buffer. remove the wash solution.
wash each well once with 1 ml of pbs. remove the wash solution.at this point the samples are ready fordna staining. if nodna stainingis desired, proceed toimagingif 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 stainingwash each well with 1 ml of pbs. remove the wash solution.
repare 1xhoechst 33342 solutionby diluting stock solution ofhoechst 333421:2000. the final concentration of 1xhoechst 33342 solutionis 5 µg/ml.final concentrations of 1xhoechst 33342may range from 2 μg/ml to 10 μg/ml.
add 1 ml of 1xhoechst 33342 solutionper well.protect from light. incubate for 30 minutes at room temperature.
remove the hoechst 33342 solution.
wash each well twice with 1 ml of pbs.
remove the wash solution.
imaginglabeled cells are compatible with all methods of slide preparation
selected references: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.
淺談自動萃取器
真空管式爐的作用
直埋保溫無縫管詳細參數
二手噴霧干燥制粒機在制藥廠食品廠的應用
MT/T748-2007工業型煤冷壓強度測試方法
Clickchemistrytools-HPG/AHA Protein Synthesis Assay Protocol
環馳軸承的發展歷程
FQ641F46氣動釜底襯氟放料球閥的用途和工作原理
袋式過濾器損壞的原因
特高壓避雷器監測器測試儀
工業除濕機在食品生產車間倉庫中的重要作用
應該如何選擇除塵設備
PCR定量試劑盒在藥物研發中的潛在應用評估
密閉式發泡結構橡塑保溫管特點講解
從這個方面竟然可以了解到油顆粒度分析儀的優勢
【時訊科普】電子天平專業術語
手動啟閉機的在安裝時要注意什么
數字便攜式相位伏安表產品用途特點及參數
如何讀懂預包裝食用植物油標簽并判定好壞食用油品質檢測儀
黑色5月！行業變局下，18家醫藥企業高管離職