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Tandem Purification of His6-3x FLAG Tagged Proteins for Mass Spectrometry from Arabidopsis
串联纯化用于质谱分析的拟南芥His6-3x FLAG标记蛋白   

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Abstract

Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His6-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and specificity of our methods allow for purification of proteins expressed at native levels from endogenous promoters to enable uncovering the functional roles of plant protein complexes.

Keywords: Tandem affinity purification(串联亲和纯化), Mass spectrometry(质谱法), Arabidopsis thaliana(拟南芥), Protein purification(蛋白纯化)

Background

Protein complexes function as signaling platforms, molecular machines, and scaffolds upon which cellular life is built. Identification of protein-protein interaction (PPI) or the composition of a protein complex is of enormous importance to provide insight into the biochemical function of genes. Therefore, facile and robust methods for discovering PPI are required to understand how genotype determines phenotype in plants.
   Commonly used protein purification methods involve chromatography, such as size-exclusion chromatography (also known as gel filtration), ion exchange chromatography and affinity chromatography. Combining several different chromatographic approaches is often required to reach sufficient purity to identify relevant complexes. Recently, affinity purification coupled with mass spectrometry (AP-MS) has emerged as a powerful biochemical approach of systematically identifying in vivo protein-protein associations. By fusing one (for one-step AP-MS) or two (for tandem AP-MS) affinity tags to a protein that serves as a bait, one can simultaneously isolate the bait protein and co-purify any proteins directly or indirectly associated with the bait from crude protein extracts. One-step AP-MS method can suffer a high false positive rate because of many sticky or highly abundant contaminating proteins. The tandem AP-MS method leverages an additional affinity purification step to increase selectivity, further filter out contaminants, and simultaneously enrich for captured complexes. Several affinity tags have been widely used, such as the FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) and polyhistidine (e.g., His6), each of which have different binding capacity and methods of elution, resulting in different yields and purity (Lichty et al., 2005). It is noteworthy that AP-MS has its limitations, such as different tags or combinations may lead to distinct group of non-specific interactors (false positives), long protocols may lead to loss of weakly associate partners (false negatives), and AP-MS cannot distinguish direct from indirect interaction. For more information on comparing different PPI techniques, as well as challenges or limits of those approaches, we would like to point to several in-depth reviews (Van Leene et al., 2008; Fukao, 2012; Braun et al., 2013; LaCava et al., 2015).
   We have developed a methodology for tandem-affinity purification for use in plants that is designed for the specific identification of protein complexes by mass spectrometry (Huang et al., 2016a and 2016b). We fused a His6-3x FLAG tandem affinity tag to the C-terminus of the bait protein, transformed the construct expressing the fusion protein into the model plant Arabidopsis thaliana by the floral dipping method (Zhang et al., 2006), and conducted tandem AP-MS to identify associated proteins using this methodology. The pB7HFC3.0 construct we used for cloning and expressing the fusion protein has been described (Huang et al., 2016a; 2016b; 2016c). We choose the FLAG purification over other affinity purification methods as the first purification step. This is because FLAG antibodies have low background in Arabidopsis thaliana, and by applying excessive free FLAG peptides, we can elute off FLAG-tagged proteins from beads in gentle, non-denaturing conditions. We then used Cobalt beads to bind His6-tagged proteins present in the FLAG eluates since the high binding capacity of Cobalt beads facilitates the enrichment, FLAG elution peptide removal, and further cleanup of all His6-tagged proteins from the first purification step. The tandem AP-MS protocol using the His6-3x FLAG tag was previously shown able to be sensitive and selective enough to identify protein complexes expressed at near native levels in Arabidopsis thaliana seedlings (Huang et al., 2016a).

Materials and Reagents

  1. 125 mm Qualitative Whatman paper (GE Healthcare, catalog number: 1001125 )
  2. 2 ml Seal-Rite microcentrifuge tubes (USA Scientific, catalog number: 1620-2700 )
  3. P1000 pipet tip
  4. 50 ml centrifuge tubes (VWR, catalog number: 89039-658 )
  5. Oak Ridge centrifuge tubes, 50 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3118-0050 )
  6. 15 ml conical tubes (VWR, catalog number: 89039-666 )
  7. 0.45 µm PVDF syringe filters with luer lock (EMD Millipore, catalog number: SLHVM33RS )
  8. Serological pipets
  9. 1.5 ml tube
  10. 1.7 ml Low Retention microtubes (PHENIX Research Products, catalog number: MAX-715L )
  11. 0.22 µm PVDF syringe filters with luer lock (EMD Millipore, catalog number: SLGV033RS )
  12. 15 cm plates (VWR, catalog number: 25384-326 )
  13. 30 ml syringe (BD, Luer-LokTM, catalog number: 302832 )
  14. 5 ml syringe (BD, catalog number: 305855 )
  15. 3.2 mm stainless steel balls (Bio Spec Products, catalog number: 11079132ss )
  16. Arabidopsis seedlings (Col-0 ecotype) containing transgenes that express His6-3x FLAG epitope tagged proteins (Huang et al., 2016a; 016b; 2016c)
  17. Liquid N2 for flash freezing samples
  18. Dry Ice to maintaining samples cold
  19. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 10837091001 )
  20. Isopropanol
  21. Protease inhibitor tablets, EDTA-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88266 )
  22. Phosphatase inhibitor cocktail 2 (Sigma-Aldrich, catalog number: P5726 )
  23. MG-132 (Peptides International, catalog number: IZL-3175-v )
  24. Phosphatase inhibitor 3 (Sigma-Aldrich, catalog number: P0044 )
  25. Dynal Talon Magnetic beads (Thermo Fisher Scientific, NovesTM, catalog number: 10104D )
  26. M2 anti-FLAG antibody (Sigma-Aldrich, catalog number: F1804 )
  27. Dynal Protein G Magnetic beads (Thermo Fisher Scientific, NovesTM, catalog number: 10003D )
  28. 3x FLAG elution buffer
  29. 3x FLAG peptide (Sigma-Aldrich, catalog number: F4799 )
  30. Murashige and Skoog medium (MP BIOMEDICALS, catalog number: 092610024 )
  31. Agar (Sigma-Aldrich, catalog number: A1296 )
  32. Sodium phosphate monobasic dehydrate (NaH2PO4·H2O) (Sigma-Aldrich, catalog number: 71505 )
  33. Sodium phosphate dibasic dehydrate (Na2HPO4·H2O) (Sigma-Aldrich, catalog number: 71662 )
  34. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  35. EDTA (Sigma-Aldrich, catalog number: EDS )
  36. EGTA (Sigma-Aldrich, catalog number: E3889 )
  37. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  38. Ammonium bicarbonate (Sigma-Aldrich, catalog number: A6141 )
  39. ddH2O
  40. ½x MS-agar media (see Recipes)
  41. SII buffer (store at 4 °C) (see Recipes)
  42. SII+ buffer (make fresh) (see Recipes)
  43. FLAG to His buffer (store at 4 °C) (see Recipes)
  44. Ammonium bicarbonate buffer (make fresh) (see Recipes)
  45. 3x FLAG peptide (store at -80 °C) (see Recipes)

Equipment

  1. Autoclave
  2. Retsch 400 mixer mill (Retsch, model: 400 Mixer Mill )
  3. Retsch mixer mill adapter racks for single use tubes (Retsch, catalog number: 22.008.0008 )
  4. 35 ml grinding jars with 20 mm stainless steel balls (referred as ‘ball mill’ hereafter) (Retsch, catalog numbers: 01.462.0214 , 05.368.0062 )
  5. Growth chamber for seedlings (Geneva Scientific, catalog number: CU-36L6 )
  6. Probe sonicator with microtip attachment (Thermo Fisher Scientific, Fisher Scientific, model: 505 )
  7. High speed centrifuge (≥ 20,000 x g) (Beckman Coulter, model: any Avanti J series )
  8. High speed rotor (Beckman Coulter, model: JA-20 )
  9. Microcentrifuge (Eppendorf, model: 5424 )
  10. Swinging bucket centrifuge (Eppendorf, model: 5810 R )
  11. Magnetic stand to capture beads, Dynamag-15 (Thermo Fisher Scientific, catalog number: 12301D )
  12. Magnetic stand to capture beads, Dynamag-2 (Thermo Fisher Scientific, catalog number: 12321D )
  13. Heating mixer (Eppendorf, model: 5355 )

Procedure

  1. Harvest tissue
    1. Grow Arabidopsis seedlings expressing His6-3x FLAG tagged proteins on sterilized Whatman on top of ½x MS 15 cm plates for 10-12 days under specific light conditions.
    2. For small scale experiments (approximately 0.5 g tissue per sample), collect tissue into 2~5 2 ml bullet tubes containing three 3.2 mm stainless steel balls. For large scale affinity purification/mass spectrometry (AP/MS), harvest 5 g tissue per sample in foil and harvest 3 packages (5 g) as replicates.
    3. Label with date, name of tissue, weight, growth conditions (constant light, 12 h light:12 h dark, Short Days, Long Days, constant dark, constant blue, constant red, etc.), and Zeitgeber time.
    4. Freeze in Liquid N2 and store at -80 °C.
    Note: Growing seedlings on Whatman facilitates seedling removal while minimizing transfer of growth media. For large scale affinity purification and mass spectrometry (AP/MS), put tissues in a corner of the foil and form a ball, so that it is easy to be broken up and poured into the metal ball mill.

  2. Grind tissue using the Retsch 400 mixer mill
    1. Put Retsch mixer mill adapter racks (for 2 ml tubes) or the 35 ml grinding Jar which contains one 20 mm stainless steel ball (ball mill) into Liquid N2 to cool before use. Carefully put 5 g frozen tissue into the 35 ml grinding Jars using a pre-cooled spatula.
    2. Break up tissue by gently inverting the ball mill (large scale) or by using pre-cooled P1000 tip to crunch tissue in bullet tubes (small scale).
    3. For small scale, grind samples either 4 times at 30 Hz for 45 sec for bullet tubes. For large scale using the ball mill, first grind at 25 Hz for 45 sec to create cushion of tissue in ball mill to prevent damage, then 4 times at 30 Hz for 45 sec.
    4. Cool the ball mill in Liquid N2 between grinding. For 2 ml bullet tubes, open caps once to release pressure inside the tube between each grinding. Also scrape compacted sample off lid with pre-cooled pipet tip to dislodge. During handling, keep grinding jars, adapter racks, holders and tubes in Liquid N2 and work quickly to prevent samples from thawing.
    5. When finished, transfer bullet tubes to dry ice (open once to release pressure), for ball mill transfer powder to 50 ml conical on dry ice.
    6. Continue to the purification steps, or store at -80 °C for up to one month.
    Notes:
    1. When handling samples: Use Liquid N2 and dry ice to keep everything cool. Label 50 ml Falcon conical tubes (on both cap and side) for the ground powder (large scale) and put them on dry ice. The grinding process takes ~1 h with clean up, so we grind multiple samples one day before the IP.
    2. If you will go directly from grinding samples into the prep, before grinding preps you should make fresh 100 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol, add protease inhibitor tablet to buffer SII and thaw the phosphatase inhibitor 2 and MG-132 at RT, (at 4 °C it will freeze again). Do not add into the buffer until you grind tissue into powders. Put all 15 ml, 50 ml Falcon conical tubes, 50 ml centrifugation tubes, long pipet tips, tips with filter into the cold room to cool before use. Put the cross-linked anti-FLAG beads on a roller to fully resuspend (use the magnetic stand to help get beads off the bottom, or just tapping the tube bottom with fingers).

  3. Resuspend ground tissue
    1. Make up SII+ buffer with protease inhibitor tablet (1 mini tablet for 10 ml or 1 tablet up to 50 ml), 1 mM PMSF (100x stock freshly made in isopropanol), 1x phosphatase inhibitors 2&3 (from 100x stocks), 50 µM MG-132 (from 200x,10 mM stock). 30 ml is enough for two packages of 5 g tissue.
    2. Add 1 packed tissue volume of SII+ (typically 500~800 µl for 0.5 g of tissue for small scale or ~12-14 ml for 5 g of tissue) to ground tissue and rotate at 4 °C for 10 min. Do not vortex (you can put a rotator onto a shaker, so that it is resuspended gently).
      Note: During this time, label 1.5 ml tubes for quality control steps: Input before FLAG IP/total extraction, FLAG IP flow through, FLAG beads, FLAG elution 1-4 (E1-E4), FLAG elution combined/His IP input, His IP flow through, His beads/Talon beads as well as washes if you want to save. Three low retention tubes are needed for each sample, and they are for: FLAG IP transfer, FLAG IP combined elutes, Talon Dynal beads (His beads, final tube, label well).

  4. Sonicate
    1. Sonicate resuspension twice at 40% amplitude (power) for 20 sec, with 1 sec on/off pulse. Keep the sample tubes on ice between each sonication. For small scale preparations, move resuspended tissue to a new tube to avoid damaging the sonicator tip with the stainless steel beads used for tissue disruption in Liquid N2.
      Note: Wear earplugs to protect your ears. Let the sample sit on ice to cool before moving on to the next sonication. Move the 50 ml tubes up and down while sonicating so that the microtip will thoroughly break up any chunks. Also, precool the centrifuge and rotor to 4 °C at this time.

  5. Clarify extract
    1. Spin clarify the samples at ≥ 20,000 x g, 10 min, 4 °C, twice.
    2. Filter the clarified supernatant to remove any chunks with a 0.45 µm filter attached to syringe of adequate size (e.g., 30 ml syringe). The filtered extract goes into a 15 ml conical tube. Note volume.
    3. Save 90 µl for input control.
    4. Measure protein concentration. Usually for 5 g 10-day-old Arabidopsis seedlings, we had a concentration of 5-10 mg/ml. So for large scale, we use a total protein of 75-150 mg.

  6. Pre-wash the crosslinked anti-FLAG Dynal beads
    1. During the second centrifugation, add 900 µl SII buffer without supplements in a tube.
    2. Add 250 µl of crosslinked anti-FLAG Dynal beads (we calculated ~5 µl beads/1 mg of extract [e.g., 250 µl for 5 g tissues]) into the liquid, pipet several times to wash the tip so that all beads go into the solution.
    3. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    4. Put beads on magnetic stand Dynamag-2, and wait 1 min for beads being separated from supernatant.
    5. Remove supernatant.
    6. Wash once more with 900 µl SII buffer without supplements, then remove supernatant and add 400 µl SII+ buffer. Keep pre-washed beads sit on ice.
    Note: Crosslink 2 µg of M2-FLAG antibody per 60 µl of Protein G beads following instructions in the manual for Dynal Protein G beads.

  7. Begin incubation with anti-FLAG Dynal beads
    1. When ready to incubate beads with extract, put beads on magnetic stand and wait 1 min.
    2. Remove supernatant.
    3. Pipet ~500 µl of extract onto the beads, resuspend, and pipet back all the beads into the 15 ml conical tube, repeat 2 more time to transfer all the beads.
    4. Start the immunoprecipitation (IP) on a rotator for 30 min-1 h, 4 °C.
    Note: Preparation during FLAG-IP: prep the 3x FLAG elution buffer. To make 500 µg/ml 3x FLAG peptide in the FLAG to His buffer, pipet 54.5 µl of 33 mg/ml 3x FLAG stock solution into 3.6 ml buffer (3.6 ml gives a little extra volume for two samples’ elution).

  8. Bead capture, washes, and transfer to 1.5 ml low retention tube
    1. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    2. Place tubes on magnetic rack Dynamag-15. Wait 2 min.
    3. Remove flow through without disturbing beads. Stick a P1000 pipet tip on end of 14 ml serological pipet to control flow for large scale capture.
    4. Save the flow through for controls.
    5. Wash beads in 10 ml SII buffer (no supplements needed) (≥ 20 bead volumes, so at least 5 ml for large scale or 1 ml for small scale).
    6. Rotate for 5 min.
    7. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    8. Place tubes on magnetic rack Dynamag-15. Wait 2 min.
    9. Remove Wash without disturbing beads. Save for controls.
    10. Wash beads in 10 ml SII buffer (no supplements needed).
    11. Rotate for 1 min.
    12. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    13. Place tubes on magnetic rack. Wait 2 min.
    14. Remove Wash without disturbing beads. Save for controls.
    15. On the third wash, wash beads off wall with 900 µl FLAG to His buffer.
    16. Transfer beads to a 1.5 ml low retention tube labeled as FLAG IP transfer.
    17. Place tubes on magnetic stand Dynamag-2. Wait 1 min.
    18. Remove Wash without disturbing beads.
    19. Transfer any remaining beads from 15 ml conical to 1.5 ml tube in 900 µl FLAG to His buffer.
    20. Place tubes on magnetic stand Dynamag-2. Wait 1 min.
    21. Remove Wash without disturbing beads.
    22. Repeat washing with 900 µl FLAG to His buffer two more times.
    Note: On the third wash, control volume so that you can transfer all beads from 15 ml conical tube to 1.5 ml low retention tube to do the following washes. The FLAG to His buffer has no EDTA/EGTA and less detergent, so it is compatible with Cobalt/Nickel NTA beads.

  9. Elution off the beads
    1. Remove supernatant off beads.
    2. Add 400 µl elution buffer made earlier (FLAG to His buffer + 500 µg/ml [ng/µl] 3x FLAG peptide).
    3. Rotate beads for 15 min at 4 °C.
    4. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    5. Place tubes on magnetic stand Dynamag-2. Wait 1 min.
    6. Remove elution without disturbing beads, transfer 1/10 vol to elution 1 (E1) sample tubes, rest to low retention protein tube labeled FLAG IP combined elutes.
    7. Repeat elution at 4 °C for 15 min, save the same amount from the second elution for quality control and put the rest of the 2nd elution to low retention protein tube labeled FLAG IP combined elutes.
    8. Elute another two times with 400 µl elution, rotate beads at 30 °C for 15 min. Save 1/10 of each elution for quality controls and combine the rest of the 3rd and 4th elution to the low retention protein binding tube.
    9. Mix combined elution tube. Remove 1/20 volume to tube for analysis.
    Note: During the 2nd~3rd elution, pre-wash Talon Dynal beads (His beads) with FLAG to His buffer (No EDTA or EGTA, since it will strip cobalt off the resin). Use at least ~1/5 volume of beads that you used in FLAG IP, but can go higher if you observe protein in Talon bead flow through. Also, make ≥ 5 ml 25 mM ammonium bicarbonate buffer, prepared fresh. Filter through a 0.22 µm filter.

  10. His IP
    1. Transfer combined eluates to low protein binding tube with washed Talon beads.
    2. Incubate for 20 min, at 4 °C with rotation.
    3. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    4. Place tubes on magnetic stand Dynamag-2. Wait 1 min.
    5. Remove Wash without disturbing beads.
    6. Wash with 900 µl FLAG to His buffer by mixing by gently inverting the tube.
    7. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps. Then repeat the wash one more time.
    8. Remove the 2nd FLAG to His wash without disturbing beads. Then wash beads with 900 µl 25 mM ammonium bicarbonate buffer and gently invert the tube. Repeat twice as previous washes.
    9. During the last wash with 900 µl 25 mM ammonium bicarbonate buffer, once the beads are completely resuspended by gently inverting the tube, remove 1/10 (90 µl) volume to new tube for quality control.
    10. Spin tubes for 1 min at ≥ 1,000 x g to collect solution from caps.
    11. Place tubes on magnetic stand Dynamag-2. Wait 1 min and then remove all of wash without disturbing beads.
    12. Flash freeze beads in Liquid N2, store in -80 °C.
    13. After His IP, running a Western blot or silver staining to check the quality of the affinity purification. For example, load on 10% SDS-PAGE gel of combined FLAG eluates (serves as an input for His purification), 10% of flow-through/unbound for the His purification, and 10% of His beads after binding. This is to test if the bait protein is well enriched after His purification. A good practice is to also compare protein purifications of your protein of interest to control purifications (using a His6-3x FLAG tagged control protein such as Green Fluorescent Protein) by silver stain (Chevallet et al., 2006) to identify unique bands associated with your protein of interest. Submit protein/beads complex for digestion and sequencing at mass spectrometry facility.

Data analysis

Two to four independent biological replicate affinity purifications should be done to determine reproducibility of mass spectrometry identifications from purifications, as mentioned previously (Huang et al., 2016a and 2016b). All epitope-tagged lines should be checked for functionality prior to use, preferably by complementing characterized mutants (Huang et al., 2016a).

Notes

  1. Wear and change gloves often. The top identified contaminating proteins are keratin and collagen from humans. Do not touch clothes with gloved hands. All the buffers, tips, filters, syringes are kept in cold room and separated from others, and for MS-use only.
  2. Other than when specified, all the work should be done in the cold room with ice bucket.
  3. The use of non-carbohydrate based resins, such as the polystyrene paramagnetic Dynal beads, will reduce background from plant tissues.
  4. Refer to the resin manufactures specifications for information about buffer, salt and detergent compatibility.

Recipes

  1. ½x MS-agar media (1 L)
    2.205 g Murashige and Skoog medium
    7 g agar
    Autoclave, and dispense ~40 ml per 15 cm dish
  2. SII buffer, store at 4 °C
    100 mM Na-Phosphate, pH 8.0
    150 mM NaCl
    5 mM EDTA
    5 mM EGTA
    0.1% Triton X-100
    Filter through a 0.22 µm filter to sterilize
  3. SII+ buffer (make fresh)
    Note: Add following supplements to SII buffer above just before use.
    1 mM phenylmethylsulfonyl fluoride
    1x protease inhibitor mix
    1x phosphatase inhibitor II
    1x phosphatase inhibitor III
    50 µM MG-132
  4. FLAG to His buffer (store at 4 °C)
    100 mM Na-Phosphate, pH 8.0
    150 mM NaCl
    0.05% Triton X-100
    Filter through a 0.22 µm filter to sterilize
  5. Ammonium bicarbonate buffer (make fresh)
    25 mM ammonium bicarbonate in ddH2O, made fresh, then filter through a 0.22 µm filter to sterilize
  6. 3x FLAG peptide (store at -80 °C)
    ≥ 33 mg/ml MDYKDHDGDYKDHDIDYKDDDDK resuspended in phosphate buffer, pH 8.0

Acknowledgments

The Nusinow lab acknowledges support by the National Science Foundation (IOS 1456796). This protocol was adapted from Huang et al., 2016a.

References

  1. Braun, P., Aubourg, S., Van Leene, J., De Jaeger, G. and Lurin, C. (2013). Plant protein interactomes. Annu Rev Plant Biol 64: 161-187.
  2. Chevallet, M., Luche, S. and Rabilloud, T. (2006). Silver staining of proteins in polyacrylamide gels. Nat Protoc 1(4): 1852-1858.
  3. Fukao, Y. (2012). Protein-protein interactions in plants. Plant Cell Physiol 53(4): 617-625.
  4. Huang, H., Alvarez, S., Bindbeutel, R., Shen, Z., Naldrett, M. J., Evans, B. S., Briggs, S. P., Hicks, L. M., Kay, S. A. and Nusinow, D. A. (2016a). Identification of evening complex associated proteins in Arabidopsis by affinity purification and mass spectrometry. Mol Cell Proteomics 15(1): 201-217.
  5. Huang, H., Alvarez, S. and Nusinow, D. A. (2016b). Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS). Data Brief 8: 56-60.
  6. Huang, H., Yoo, C. Y., Bindbeutel, R., Goldsworthy, J., Tielking, A., Alvarez, S., Naldrett, M. J., Evans, B. S., Chen, M. and Nusinow, D. A. (2016c). PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis. Elife 5: e13292.
  7. LaCava, J., Molloy, K. R., Taylor, M. S., Domanski, M., Chait, B. T. and Rout, M. P. (2015). Affinity proteomics to study endogenous protein complexes: pointers, pitfalls, preferences and perspectives. Biotechniques 58(3): 103-119.
  8. Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J. and Tan, S. (2005). Comparison of affinity tags for protein purification. Protein Expr Purif 41(1): 98-105.
  9. Van Leene, J., Witters, E., Inze, D. and De Jaeger, G. (2008). Boosting tandem affinity purification of plant protein complexes. Trends Plant Sci 13(10): 517-520.
  10. Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W. and Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-646.

简介

串联亲和纯化是识别与已知感兴趣基因相关联的蛋白质复合物的有力方法。 该方案描述了一种用于捕获用His6-3x FLAG标记的蛋白质的方法,用于通过质谱法进行珠粒消化和鉴定。 我们的方法的高灵敏度和特异性允许从内源启动子纯化以天然水平表达的蛋白质,以揭示植物蛋白复合物的功能作用。
【背景】蛋白质复合物作为信号平台,分子机器和构建细胞生命的支架。鉴定蛋白质 - 蛋白质相互作用(PPI)或蛋白质复合物的组成对于提供对基因生物化学功能的了解是非常重要的。因此,需要了解发现PPI的简便方法,以了解基因型如何决定植物中的表型。
通常使用的蛋白质纯化方法包括色谱法,例如大小排阻色谱法(也称为凝胶过滤),离子交换层析和亲和层析。通常需要组合几种不同的色谱方法以达到足够的纯度以鉴定相关的复合物。最近,亲和纯化与质谱联用(AP-MS)已经成为系统地鉴定体内蛋白质 - 蛋白质关联的强大的生物化学方法。通过将一个(用于一步AP-MS)或两个(用于串联AP-MS)亲和标签融合到用作诱饵的蛋白质,可以同时分离诱饵蛋白质并共同纯化直接或间接与来自粗蛋白提取物的诱饵。由于许多粘稠或高度丰富的污染蛋白质,一步AP-MS方法可能遭受高假阳性率。串联AP-MS方法利用额外的亲和纯化步骤来增加选择性,进一步过滤掉污染物,并同时富集捕获的复合物。已经广泛使用几种亲和标签,例如FLAG肽(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)和多组氨酸(例如His6),其各自具有不同的结合能力和洗脱方法,导致不同的产率和纯度(Lichty等人,2005)。值得注意的是,AP-MS有其局限性,例如不同的标签或组合可能导致不同组的非特异性交互作用(假阳性),长协议可能导致弱关联伙伴(假阴性)的丧失,AP- MS不能区分直接和间接的相互作用。有关比较不同PPI技术以及这些方法的挑战或限制的更多信息,我们想指出几个深入的评论(Van Leene et al。,2008; Fukao,2012; Braun et al。,2013; LaCava等,2015)。
我们已经开发了一种串联亲和纯化方法,用于通过质谱法特异性鉴定蛋白质复合物的植物中(Huang等,2016a和2016b)。我们将His6-3x FLAG串联亲和标签融合到诱饵蛋白的C末端,通过花浸法将表达融合蛋白的构建体转化成拟南芥拟南芥(Zhang et al。,2006),并进行串联AP-MS使用该方法鉴定相关蛋白。已经描述了用于克隆和表达融合蛋白的pB7HFC3.0构建体(Huang等,2016a; 2016b; 2016c)。我们选择FLAG纯化比其他亲和纯化方法作为第一个纯化步骤。这是因为FLAG抗体在拟南芥中具有较低的背景,并且通过施用过量的游离FLAG肽,我们可以在温和,非变性的条件下从珠中洗出FLAG标记的蛋白质。然后,我们使用钴珠结合存在于FLAG洗脱液中的His6标记的蛋白质,因为钴珠的高结合能力促进了富集,FLAG洗脱肽的去除以及从第一个纯化步骤进一步清除所有His6标记的蛋白质。先前证明使用His6-3x FLAG标签的串联AP-MS方案能够具有敏感性和选择性,足以鉴定拟南芥幼苗中接近原生水平表达的蛋白质复合物(Huang et al。,2016a)。

关键字:串联亲和纯化, 质谱法, 拟南芥, 蛋白纯化

材料和试剂

  1. 125mm定性Whatman纸(GE Healthcare,目录号:1001125)
  2. 2ml Seal-Rite微量离心管(USA Scientific,目录号:1620-2700)
  3. P1000移液器吸头
  4. 50ml离心管(VWR,目录号:89039-658)
  5. Oak Ridge离心管,50ml(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3118-0050)
  6. 15ml锥形管(VWR,目录号:89039-666)
  7. 0.45μm具有路厄锁的PVDF针筒过滤器(EMD Millipore,目录号:SLHVM33RS)
  8. 血清移液器
  9. 1.5 ml管
  10. 1.7ml低保留微管(PHENIX Research Products,目录号:MAX-715L)
  11. 0.22微米PVDF注射器过滤器(EMD Millipore,目录号:SLGV033RS)
  12. 15cm板(VWR,目录号:25384-326)
  13. 30ml注射器(BD,Luer-Lok TM ,目录号:302832)
  14. 5ml注射器(BD,目录号:305855)
  15. 3.2mm不锈钢球(Bio Spec Products,目录号:11079132ss)
  16. 包含表达His 6 -6-FLAG表位标签蛋白的转基因的拟南芥(Arabidopsis)苗(Col-0生态型)(Huang等人,2016a; 016b ; 2016c)
  17. 液体N <2>用于快速冷冻样品
  18. 干冰保持样品冷却
  19. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:10837091001)
  20. 异丙醇
  21. 蛋白酶抑制剂片剂,无EDTA(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88266)
  22. 磷酸酶抑制剂混合物2(Sigma-Aldrich,目录号:P5726)
  23. MG-132(Peptides International,目录号:IZL-3175-v)
  24. 磷酸酶抑制剂3(Sigma-Aldrich,目录号:P0044)
  25. Dynal Talon磁珠(Thermo Fisher Scientific,Noves TM ,目录号:10104D)
  26. M2抗FLAG抗体(Sigma-Aldrich,目录号:F1804)
  27. Dynal Protein G磁珠(Thermo Fisher Scientific,Noves TM ,目录号:10003D)
  28. 3x FLAG洗脱缓冲液
  29. 3x FLAG肽(Sigma-Aldrich,目录号:F4799)
  30. Murashige和Skoog培养基(MP BIOMEDICALS,目录号:092610024)
  31. 琼脂(Sigma-Aldrich,目录号:A1296)
  32. 磷酸二氢钠一水合物(NaH 2 PO 4·H 2 O)(Sigma-Aldrich,目录号:71505)
  33. 磷酸二氢钠二水合物(Na 2 HPO 4·H 2 O)(Sigma-Aldrich,目录号:71662)
  34. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  35. EDTA(Sigma-Aldrich,目录号:EDS)
  36. EGTA(Sigma-Aldrich,目录号:E3889)
  37. Triton X-100(Sigma-Aldrich,目录号:T9284)
  38. 碳酸氢铵(Sigma-Aldrich,目录号:A6141)
  39. ddH sub 2 O
  40. ½xMS-琼脂培养基(参见配方)
  41. SII缓冲液(储存在4°C)(参见配方)
  42. SII +缓冲液(新鲜)(参见配方)
  43. FLAG到他的缓冲液(存储在4°C)(参见配方)
  44. 碳酸氢铵缓冲液(新鲜)(参见配方)
  45. 3x FLAG肽(储存在-80℃)(参见配方)

设备

  1. 高压灭菌器
  2. Retsch 400混合机(Retsch,型号:400 Mixer Mill)
  3. Retsch混合机适配器架一次性管(Retsch,目录号:22.008.0008)
  4. 35ml具有20mm不锈钢球(以下称为"球磨机")的研磨罐(Retsch,目录号:01.462.0214,05.368.0062)
  5. 幼苗生长室(Geneva Scientific,目录号:CU-36L6)
  6. 带有微尖附件的探头超声波仪(Thermo Fisher Scientific,Fisher Scientific,型号:505)
  7. 高速离心机(≥20000xg)(Beckman Coulter,型号:任何Avanti J系列)
  8. 高速转子(Beckman Coulter,型号:JA-20)
  9. 微量离心机(Eppendorf,型号:5424)
  10. 摆动离心机(Eppendorf,型号:5810R)
  11. 磁性支架捕获珠,Dynamag-15(Thermo Fisher Scientific,目录号:12301D)
  12. 磁性支架捕获珠,Dynamag-2(Thermo Fisher Scientific,目录号:12321D)
  13. 加热混合器(Eppendorf,型号:5355)

程序

  1. 收获组织
    1. 在特异光条件下,在灭菌的Whatman上,在1/2xMS 15cm板的顶部上生长表达His 6 - 3x FLAG标记蛋白的拟南芥幼苗10-12天。
    2. 对于小规模实验(每个样品约0.5g组织),将组织收集到含有三个3.2mm不锈钢球的2〜52ml子弹管中。对于大规模亲和纯化/质谱(AP/MS),收获5克组织每个样品在箔和收获3包(5克)作为重复。
    3. 用日期,组织名称,体重,生长条件(恒定光,12小时光:12小时黑暗,短日,长日,恒定黑暗,恒定蓝色,恒定红色,等等)标记标签。和Zeitgeber时间
    4. 在液氮中冻结并储存在-80℃。
    注意:在Whatman上种植幼苗有助于移除幼苗,同时使生长培养基的转移最小化。对于大规模亲和纯化和质谱(AP/MS),将组织放置在箔的角落中并形成球,使得其易于破碎并倒入金属球磨机中。

  2. 使用Retsch 400混合磨粉机磨碎组织
    1. 将Retsch混合机研磨机适配器架(用于2ml管)或含有一个20mm不锈钢球(球磨机)的35ml研磨罐放入Liquid N 2中以在使用前冷却。使用预冷刮刀小心地将5g冷冻组织放入35ml研磨罐中
    2. 通过轻轻颠倒球磨机(大规模)或使用预冷却的P1000尖端来破碎组织,以在子弹管(小规模)中破碎组织。
    3. 对于小规模,在30Hz下研磨样品4次,45秒用于子弹管。对于大规模使用球磨机,首先在25Hz下研磨45秒以在球磨机中产生组织垫以防止损坏,然后在30Hz下4次,45秒。
    4. 在研磨之间在N/2液中冷却球磨机。对于2ml子弹管,打开盖子一次,以在每次研磨之间释放管内的压力。还用预冷却的移液管尖端刮掉盖上的压实样品,以移出。在处理过程中,保持研磨罐,适配器架,支架和管在Liquid N <2>中,并迅速工作以防止样品解冻。
    5. 完成后,将子弹管转移到干冰(打开一次以释放压力),用于球磨转移粉末到50毫升在干冰上的锥形。
    6. 继续纯化步骤,或在-80℃下储存长达一个月。
    注意:
    1. 处理样品时:使用Liquid N 2和干冰保持一切凉爽。标记50ml研磨粉末(大规模)的Falcon锥形管(在盖和侧面上),并将其放在干冰上。研磨过程需要〜1小时的清理,所以我们在IP前一天研磨多个样品。
    2. 如果你将直接从研磨样品进入制备,在研磨制备前,你应该在异丙醇中制备新鲜的100mM苯甲基磺酰氟(PMSF),添加蛋白酶抑制剂片剂缓冲SII和解冻磷酸酶抑制剂2和MG-132 RT,(在4℃下它将再次冻结)。不要加入缓冲液,直到你把组织粉碎为止。将所有15 ml,50 ml Falcon锥形管,50 ml离心管,长移液管吸头,带过滤器的吸头放入冷室,使用前冷却。将交联的抗FLAG珠子放在滚筒上以完全重悬(使用磁性支架帮助从底部取下珠子,或用手指轻轻敲击管子底部)。

  3. 重悬地面组织
    1. 用蛋白酶抑制剂片剂(1微量片剂用于10ml或1片剂直到50ml),1mM PMSF(100×在异丙醇中制备的储备液),1×磷酸酶抑制剂2和3(来自100×储备液),50μM MG-132(来自200x,10mM储备液)。 30毫升是足够的两个包装5克的组织
    2. 添加1包装组织体积的SII +(通常500〜800微升0.5克组织小规模或〜12-14毫升5克组织)到地面组织,并在4℃下旋转10分钟。不要涡旋(你可以把旋转器放在振动器上,以便轻轻地重新悬浮)。
      注意:在此期间,标记1.5ml管用于质量控制步骤:在FLAG IP /总提取之前输入,FLAG IP流过,FLAG珠,FLAG洗脱1-4(E1-E4),FLAG洗脱组合/His IP输入,他的IP流通,他的珠/Talon珠以及洗涤,如果你想保存。每个样品需要三个低保留管,它们用于:FLAG IP转移,FLAG IP组合洗脱,Talon Dynal珠(His珠,最终管,标记孔)。
  4. 超声
    1. 以40%振幅(功率)超声重悬两次20秒,1秒开/关脉冲。在每次超声处理之间保持样品管在冰上。对于小规模制备,将重新悬浮的组织移至新管,以避免用用于液体N sub 2中的组织破坏的不锈钢珠损坏超声波仪尖端。
      注意:佩戴耳塞以保护耳朵。让样品在冰上冷却,然后移动到下一个超声处理。在超声处理的同时,上下移动50 ml试管,使微尖完全破碎任何块。此外,此时将离心机和转子预冷至4°C。

  5. 澄清提取物
    1. 旋转澄清样品在≥20,000 x em ,10分钟,4℃,两次。
    2. 过滤澄清的上清液以除去任何具有0.45μm过滤器的块,该过滤器连接到足够大小的注射器(例如,30ml注射器)。过滤的提取物进入15ml锥形管。音量。
    3. 保存90μl输入控制。
    4. 测量蛋白浓度。通常对于5g 10日龄拟南芥幼苗,我们的浓度为5-10mg/ml。因此,对于大规模,我们使用75-150毫克的总蛋白
  6. 预洗交联的抗FLAG Dynal珠子
    1. 在第二次离心时,在管中加入900μl不含补充物的SII缓冲液
    2. 加入250μl交联的抗-FLAG Dynal珠(我们计算〜5μl珠/1mg提取物[例如对于5g组织为250μl])至液体中,移液管数次洗涤尖端,使所有珠子进入溶液
    3. 旋转管1分钟,≥1000x g 从帽子收集溶液
    4. 将珠子放在磁力架Dynamag-2上,等待1分钟,使珠子与上清液分离
    5. 除去上清液。
    6. 再次用900μlSII缓冲液(不含补充剂)洗涤一次,然后除去上清液,加入400μlSII +缓冲液。保持预洗的珠子坐在冰上。
    注意:按照Dynal Protein G珠子手册中的说明,每60μl蛋白G珠子交联2μgM2-FLAG抗体。

  7. 开始与抗-FLAG Dynal小珠孵育
    1. 当准备好用提取物孵育珠子时,将珠子放在磁力架上并等待1分钟
    2. 除去上清液。
    3. 吸取〜500μl的提取物到珠子上,重悬,并将所有的珠子移回15ml锥形管,重复2次以上,以转移所有的珠子。
    4. 在旋转器上开始免疫沉淀(IP)30分钟-1小时,4℃。
    注意:在FLAG-IP中的制备:制备3x FLAG洗脱缓冲液。为了在FLAG中制备500μg/ml 3x FLAG肽至His缓冲液,将54.5μl33mg/ml 3x FLAG储备溶液移至3.6ml缓冲液(3.6ml,给出两个样品洗脱的一点额外体积)中。 >

  8. 珠捕获,洗涤,并转移到1.5毫升低保留管
    1. 旋转管1分钟,≥1000x g 从帽子收集溶液
    2. 将管放在磁力架Dynamag-15上。等待2分钟。
    3. 清除流动通过,不干扰珠。将P1000移液器吸头安装在14 ml血清移液管的末端,以控制流量进行大规模捕获
    4. 保存控制流程。
    5. 在10ml SII缓冲液(不需要补充剂)中洗涤珠子(≥20个珠子体积,因此对于大规模,至少5ml,或对于小规模,至少1ml)。
    6. 旋转5分钟。
    7. 旋转管1分钟,≥1000x g 从帽子收集溶液
    8. 将管放在磁力架Dynamag-15上。等待2分钟。
    9. 删除清洗,不要打扰珠子。保存控件。
    10. 在10 ml SII缓冲液中洗涤珠子(不需要补充剂)。
    11. 旋转1分钟。
    12. 旋转管1分钟,≥1000x g 从帽子收集溶液
    13. 将管放在磁性架上。等待2分钟。
    14. 删除清洗,不要打扰珠子。保存控件。
    15. 在第三次洗涤时,用900μlFLAG将珠子从壁上洗涤到其缓冲液中
    16. 将珠子转移到标记为FLAG IP转移的1.5ml低保留管
    17. 将管放置在磁力架Dynamag-2上。等待1分钟。
    18. 清除无干扰珠子的清洗
    19. 将任何剩余的珠子从15 ml锥形转移到1.5 ml管中,在900μlFLAG中转移到His缓冲液中
    20. 将管放置在磁力架Dynamag-2上。请等待1分钟。
    21. 删除清洗,不要打扰珠子。
    22. 重复用900μlFLAG洗涤至His缓冲液两次。
    注意:在第三次洗涤时,控制体积,以便您可以将所有珠子从15毫升锥形管转移到1.5毫升低保留管进行以下洗涤。 FLAG到其缓冲液中没有EDTA/EGTA和较少的去污剂,因此它与钴/镍NTA珠相容。

  9. 洗脱珠子
    1. 去除珠粒上的上清液。
    2. 加入400μl早期制备的洗脱缓冲液(FLAG至His缓冲液+500μg/ml [ng /μl] 3x FLAG肽)。
    3. 在4℃下将珠子旋转15分钟。
    4. 旋转管1分钟,≥1000x g 从帽子收集溶液
    5. 将管放置在磁力架Dynamag-2上。请等待1分钟。
    6. 移除洗脱,不干扰珠,转移1/10体积到洗脱1(E1)样品管,休息到低保留蛋白质管标记的FLAG IP组合洗脱物。
    7. 在4℃下重复洗脱15分钟,保留与第二次洗脱相同的量用于质量控制,并将其余的第二次洗脱置于低保留蛋白质管标记的FLAG IP组合洗脱液中。
    8. 洗脱另外两次用400μl洗脱,旋转珠在30℃下15分钟。保存每个洗脱的1/10用于质量控制,并将3个和4 th洗脱的其余部分结合到低保留蛋白结合管。
    9. 混合组合洗脱管。将1/20体积除去试管进行分析。
    注意:在第2个 〜3 洗脱,用FLAG预洗涤Talon Dynal珠(His珠)至His缓冲液(无EDTA或EGTA,因为其将从树脂上剥离钴)。使用至少〜1/5体积的珠,你用于FLAG IP,但可以走得更高,如果你观察到蛋白质在Talon珠流过。此外,使≥5毫升25mM碳酸氢铵缓冲液,新鲜制备。通过0.22μm过滤器过滤。

  10. 他的IP地址
    1. 用洗涤的Talon珠将合并的洗脱液转移到低蛋白质结合管
    2. 孵育20分钟,在4℃下旋转。
    3. 旋转管1分钟,≥1000x g 从帽子收集溶液
    4. 将管放置在磁力架Dynamag-2上。请等待1分钟。
    5. 清除无干扰珠子的清洗
    6. 通过轻轻颠倒管子混合,用900μlFLAG洗涤到His缓冲液中
    7. 在≥1000xg下旋转管1分钟以从帽收集溶液。然后重复清洗一次。
    8. 去除2 nd FLAG到他的洗涤,而不干扰珠。然后用900μl25 mM碳酸氢铵缓冲液洗涤珠子,轻轻倒转试管。重复上次洗涤两次。
    9. 在最后一次用900μl25mM碳酸氢铵缓冲液洗涤的过程中,一旦通过轻轻颠倒管子完全重悬珠子,将1/10(90μl)体积除去新管以进行质量控制。
    10. 旋转管1分钟,≥1000x g 从帽子收集溶液
    11. 将管放置在磁力架Dynamag-2上。等待1分钟,然后清除所有的洗涤,而不打扰珠子。
    12. 在N液中闪烁冷冻珠子,储存在-80℃
    13. 在其IP后,进行Western印迹或银染以检查亲和纯化的质量。例如,负载在组合的FLAG洗脱液的10%SDS-PAGE凝胶上(用作His纯化的输入),10%的流通/未结合用于His纯化,和10%的His珠结合。这是为了测试诱饵蛋白在其纯化后是否富集得很好。一个好的实践是还将感兴趣的蛋白质的蛋白质纯化与对照纯化(使用His 6 Sub-3x FLAG标记的对照蛋白质,例如绿色荧光蛋白)通过银染色(Chevallet et al。 al。,2006),以鉴定与感兴趣的蛋白质相关的独特条带。提交蛋白质/珠复合物消化和测序在质谱仪设施。

数据分析

如前所述(Huang等人,2016a和2016b),应该进行两到四次独立的生物复制亲和纯化以确定来自纯化的质谱鉴定的重复性。应当在使用之前检查所有表位标记的系的功能,优选通过补充表征的突变体(Huang等人,2016a)。

笔记

  1. 经常戴上和更换手套。顶部鉴定的污染蛋白质是来自人的角蛋白和胶原蛋白。不要用戴手套的手触摸衣服。所有的缓冲液,提示,过滤器,注射器保存在寒冷的房间,与其他人分开,并仅用于MS使用。
  2. 除了规定之外,所有的工作都应该在带有冰桶的冰箱里进行
  3. 使用非碳水化合物基树脂,例如聚苯乙烯顺磁性Dynal珠,将减少植物组织的背景。
  4. 有关缓冲液,盐和洗涤剂相容性的信息,请参阅树脂制造商规格

食谱

  1. 1/2xMS-琼脂培养基(1L) 2.205克Murashige和Skoog培养基
    7 g琼脂
    高压灭菌和分配 碳酸氢铵缓冲液(使新鲜)
    25mM碳酸氢铵,在ddH 2 O中,制成新鲜的,然后通过0.22μm过滤器过滤以灭菌
  2. 3x FLAG肽(储存在-80℃)
    ≥33 mg/ml MDYKDHDGDYKDHDIDYKDDDDK重悬浮于pH 8.0的磷酸盐缓冲液中

致谢

Nusinow实验室承认国家科学基金会(IOS 1456796)的支持。该协议改编自Huang等人,2016a。

参考文献

  1. Braun,P.,Aubourg,S.,Van Leene,J.,De Jaeger,G。和Lurin,C.(2013)。  植物蛋白相互作用体 Annu Rev Plant Biol 64:161-187。
  2. Chevallet,M.,Luche,S.和Rabilloud,T。(2006)。  聚丙烯酰胺凝胶中的蛋白质的银染色。 1(4):1852-1858。
  3. Fukao,Y。(2012)。  蛋白质 - 蛋白质相互作用在植物中。 植物细胞生理学 53(4):617-625
  4. Huang,H.,Alvarez,S.,Bindbeutel,R.,Shen,Z.,Naldrett,MJ,Evans,BS,Briggs,SP,Hicks,LM,Kay,SAand Nusinow,DA(2016a) 通过亲和纯化和质谱法。 Mol细胞 蛋白质组学15(1):201-217。
  5. Huang,H.,Alvarez,S. and Nusinow,DA(2016b)。  使用串联亲和纯化和质谱(TAP-MS)在拟南芥中与晚期复合物和PCH1鉴定蛋白质相互作用物的数据。/em> 8:56-60。
  6. Huang,H.,Yoo,CY,Bindbeutel,R.,Goldsworthy,J.,Tielking,A.,Alvarez,S.,Naldrett,MJ,Evans,BS,Chen,M.and Nusinow,DA(2016c) ; PCH1整合昼夜节律和光信号通路来控制光周期响应生长在拟南芥中。 5:e13292。
  7. LaCava,J.,Molloy,KR,Taylor,MS,Domanski,M.,Chait,BT和Rout,MP(2015)。  亲和蛋白质组学研究内源性蛋白复合物:指针,陷阱,偏好和观点 生物技术 58
  8. Lichty,JJ,Malecki,JL,Agnew,HD,Michelson-Horowitz,DJand Tan,S。(2005)。  蛋白纯化的亲和标签的比较。 Protein Expr Purif 41(1):98-105。
  9. Van Leene,J.,Witters,E.,Inze,D。和De Jaeger,G。(2008)。  提高植物蛋白复合物的串联亲和纯化。 趋势植物科学13(10):517-520。
  10. Zhang,X.,Henriques,R.,Lin,SS,Niu,QW和Chua,NH(2006)。  。 1 1 1 :641-646。
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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: © 2017,  Huang  et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huang, H. and Nusinow, D. A. (2016). Tandem Purification of His6-3x FLAG Tagged Proteins for Mass Spectrometry from Arabidopsis. Bio-protocol 6(23): e2060. DOI: 10.21769/BioProtoc.2060.
  2. Huang, H., Yoo, C. Y., Bindbeutel, R., Goldsworthy, J., Tielking, A., Alvarez, S., Naldrett, M. J., Evans, B. S., Chen, M. and Nusinow, D. A. (2016c). PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis. Elife 5: e13292.
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