欢迎您, 登录 | 注册

首页 | English

X
加载中

Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western hybridization experiments using anti-CulA or anti-Nedd8 antibodies could show the ratio of neddylated vs. deneddylated CulA. Using the deneddylation assay we could show that CsnE is the last subunit joining a 7-subunit pre-assembled CSN in vitro and only then CSN can perform cullin deneddylation by the metalloprotease subunit CsnE. This assay is a fast and non-expensive method, which visualizes enzyme activity for deneddylating proteins. It might be also useful for testing the activity of other isopeptidases removing post-translational modifications from substrates in Aspergillus nidulans (A. nidulans) or other organisms.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

In vitro Deneddylation Assay
去NEDD化修饰的体外试验

微生物学 > 微生物生物化学 > 蛋白质 > 活性
作者: Anna M. Köhler
Anna M. KöhlerAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
Bio-protocol author page: a2960
Cindy Meister
Cindy MeisterAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
Bio-protocol author page: a2961
 and Gerhard H. Braus
Gerhard H. BrausAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
For correspondence: gbraus@gwdg.de
Bio-protocol author page: a2962
Vol 6, Iss 6, 3/20/2016, 1687 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1756

[Abstract] Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western hybridization experiments using anti-CulA or anti-Nedd8 antibodies could show the ratio of neddylated vs. deneddylated CulA. Using the deneddylation assay we could show that CsnE is the last subunit joining a 7-subunit pre-assembled CSN in vitro and only then CSN can perform cullin deneddylation by the metalloprotease subunit CsnE. This assay is a fast and non-expensive method, which visualizes enzyme activity for deneddylating proteins. It might be also useful for testing the activity of other isopeptidases removing post-translational modifications from substrates in Aspergillus nidulans (A. nidulans) or other organisms.
Keywords: Deneddylation(deneddylation), COP9 signalosome (CSN)(COP9 signalosome(CSN)), DEN1(DEN1), Western hybridization(Western杂交), Nedd8(NEDD8)

[Abstract]

Materials and Reagents

  1. Recombinant protein overexpression and protein purification
    1. 1 ml cuvettes 10 x 10 x 45 mm (Sarstedt AG, catalog number: 67.742 )
    2. Chromatography columns [e.g. GSTrap (GE Healthcare, catalog number: 17-5130-01 ), HisTrap (GE Healthcare, catalog number: 17-5255-01 ), Superdex 200 HR 10/300 GL (GE Healthcare, catalog number: 17-5175-01 ), HiPrep 16/60 Sephacryl S-300 HR (GE Healthcare, catalog number: 17-1167-01 )]
    3. Amicon Ultra centrifugal filters (Merck Millipore Corporation) molecular weight cut off (MWCO) depends on protein size [e.g. 30 kDa (Merck Millipore Corporation, MWCO, catalog number: UFC903024 )]
    4. MicronSep filter paper, 0.22, 47 mm (Carl Roth GmbH + Co., catalog number: A009.1 )
    5. Escherichia coli (E. coli) strains suitable for protein overexpression (e.g. Rosetta DE3) transformed with overexpression plasmid including protein sequence of interest with N- or C-terminal Glutathione-S-transferase- (GST-) or Histidine- (His-) tag
    6. Tryptone/Peptone from Casein (Carl Roth GmbH + Co., catalog number: 8952.2 ) used as 1% solution
    7. Yeast extract (Oxoid, catalog number: LP0021 ) used as 0.5% solution
    8. Sodiumchloride (NaCl) (Carl Roth GmbH + Co., catalog number: 9265.2 ) used as 1% solution
    9. Antibiotics according to the E. coli strains used
    10. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Carl Roth GmbH + Co., catalog number: 2316.4 ) used as 1 M solution, filter sterilized
    11. 99.5% denatured Ethanol with 1% MEK (VWR International, catalog number: 85033.460 ) used as 20% solution diluted in ddH2O, filter degassed
    12. Imidazole
    13. Glutathione
    14. Lysogeny broth (LB) medium (see Recipes)
    15. Lysis buffer (see Recipes)
    16. Affinity buffer (see Recipes)
    17. Elution buffer for HisTrap (see Recipes)
    18. Elution buffer for GSTrap (see Recipes)
    19. Size exclusion buffer (SEC) (see Recipes)

  2. Preparation of protein crude extracts
    1. Paper towels
    2. Spores of Aspergillus nidulans strains
    3. Miracloth (Merck Millipore Corporation, catalog number: 475855-1R )
    4. Sodiumchloride (NaCl) (Carl Roth GmbH + Co., catalog number: 9265.2) used as 0.95 % solution
    5. Liquid nitrogen (Air Liquid)
    6. Minimal medium (see Recipes)
    7. 50x AspA (see Recipes)
    8. Trace elements (see Recipes)
    9. B* buffer (see Recipes)

  3. Deneddylation assay
    1. Gel blotting paper (Schleicher & Schuell BioScience GmbH, catalog number: 10426694 )
    2. Blotting membrane AmershamTM ProtranTM 0.45 µm NC (GE Healthcare, catalog number: 10600002 )
    3. Methanol (VWR International, catalog number: 20864.320 )
    4. 2-Propanol HiPerSolv CHROMANORM (VWR International, catalog number: 20880.320 )
    5. Page ruler prestained protein ladder (Thermo Fisher Scientific, catalog number: 26616 )
    6. Acrylamid Rotiphorese® Gel 30 (37, 5:1) (Carl Roth GmbH + Co., catalog number: 3029.1 )
    7. Sucofin skimmed milk powder (TSI GmbH, catalog number: ST00227 )
    8. α-CulA, α-Nedd8 antibodies (GenScript)
    9. Monoclonal α-Tubulin antibody (Sigma-Aldrich, catalog number: T0926 )
    10. α-Actin antibody (Novus Biologicals, catalog number: NB100-74340 )
    11. α-goat rabbit secondary antibody (Thermo Fisher Scientific, InvitrogenTM, catalog number: G21234 )
    12. α-goat mouse secondary antibody (Jackson ImmunoResearch, catalog number: 115-035-003 )
    13. p-coumaric acid (Sigma-Aldrich, catalog number: C9008-5 G ) used as 400 mM stock solution dissolved in DMSO (protect from light, store at -20 °C)
    14. Luminol (Carl Roth GmbH + Co., catalog number: 4203.1 ) used as 250 mM stock solution dissolved in DMSO (protect from light, store at -20 °C)
    15. 2-mercaptoethanol (Sigma-Aldrich, Fluka Chemika, catalog number: 63700 )
    16. Sodium dodecyl sulfate (SDS) (Carl Roth GmbH + Co., catalog number: CN30.3 ) used as 7%, 0.1%, 1%, 0.2% solutions
    17. Bromophenol blue (Sigma-Aldrich, Riedel-de Haën, catalog number: 32768 ) used as 0.3% solution
    18. Ammoniumpersulfate (APS) (Sigma-Aldrich, Fluka Analytical, catalog number: 09913-100 G ) used as 10% solution
    19. Tetramethylethylendiamine (TEMED) (Carl Roth GmbH + Co., catalog number: 2367.1 )
    20. Tween-20 (Carl Roth GmbH + Co., catalog number: 9127.1 )
    21. Tris-PUFFERAN® (Carl Roth GmbH + Co., catalog number: 4855.3 ) used as 250 mM solution
    22. Glycine (Carl Roth GmbH + Co., catalog number: 3187.3 ) used as 2.5 M solution
    23. 30% Hydrogen peroxide (H2O2) (Merck Millipore Corporation, catalog number: 822287.2500 )
    24. 3x SDS sample buffer (see Recipes)
    25. 12% SDS gels (see Recipes)
    26. 2x resolving buffer (see Recipes)
    27. 2x stacking buffer (see Recipes)
    28. 10x SDS running buffer (see Recipes)
    29. 10x transfer buffer (see Recipes)
    30. 10x TBST (Tris-Buffered Saline Tween 20) buffer (see Recipes)
    31. Blocking solution (see Recipes)
    32. Detection solution A (see Recipes)
    33. Detection solution B (see Recipes)

  4. Others (material and reagents used for more than one method)
    1. Micro tube 1.5 ml (Sarstedt AG, catalog number: 72.690.001 )
    2. Micro tube 2.0 ml (Sarstedt AG, catalog number: 72.691 )
    3. Tube 115 x 28 mm, PP (Sarstedt AG, catalog number: 62.559.001 )
    4. 5 ml test tube
    5. 2 L flasks
    6. PPCO centrifuge bottles (Slaughte, Nalgene, catalog number: 525-2316 )
    7. Oak-Ridge centrifugation tubes, 50 ml (Carl Roth GmbH + Co., catalog number: PK03.1 )
    8. 300 ml baffled flasks
    9. Funnels
    10. Crushed ice
    11. 1, 4- Dithiothreitol (DTT) (Carl Roth GmbH + Co., catalog number: 6908.2 ) used as 1 M solution
    12. trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane, E-64 protease inhibitor (Sigma-Aldrich, catalog number: E3132 ) used as 1 M stock solution
    13. Phenylmethylsulfonyl fluoride (PMSF) (Carl Roth GmbH + Co., catalog number: 6367.3 ) used as 100 mM solution in Isopropanol
    14. Double destilled water (ddH2O)
    15. Millipore water
    16. Sodium chloride (NaCl) (Carl Roth GmbH + Co., catalog number: 9265.2) used as 2 M solution
    17. Tris- hydrochloride (Tris-HCl) (Carl Roth GmbH + Co., catalog number: 9090.3 )
      Note: Prepare a series of Tris-HCl solution with different concentrations and pH including 1 M solutions with pH 6.8, 7.5, 8.0 and pH 8.8; 0.75 M solution withpH 8.8;100 mM solution with pH 8.0.
    18. Ethylenediaminetetraacetic acid (EDTA) (Carl Roth GmbH + Co.) used as 0.5 mM solution
    19. Nonoxinol-40 (NP-40) (Affymetrix, USB Corporation, catalog number: 19628 )
    20. Glycerol (Carl Roth GmbH + Co., catalog number: 3783.1 )
    21. Complete protease inhibitor cocktail (Roche Diagnostics, catalog number: 11873580001 ) (see Recipes)

Equipment

  1. Biowave DNA Llife Science Spectrophotometer (Biochrom WPA, catalog number: 80-3004-70 )
  2. Centrifuge Sorvall RC3B Plus (Thermo Fisher Scientific)
  3. Centrifuge Sorvall RC5B Plus (Thermo Fisher Scientific)
  4. Rotor Sorvall SS34 (Thermo Fisher Scientific)
  5. ÄKTAexplorer 10 (GE Healthcare, catalog number: 18-1300-00 )
  6. pH meter pH526 (Sigma-Aldrich, model: WTW )
  7. Filter and degassing system (Satorius)
  8. Vacuum pump (KNF NEUBERGER, catalog number: 2.03360238 )
  9. Sonicator Sonoplus HD2070 (Bandelin)
  10. Superloop 1/16’’ (GE Healthcare, catalog number: 18-1113-82 )
  11. Sample loop 5 ml (GE Healthcare, catalog number: 18-1140-53 )
  12. Mixer Mill MM 400 (Retsch)
  13. Vortex Genie 2 (Scientific Industries)
  14. Pipettes (1-5,000 µl) (Gilson)
  15. Centrifuge Biofuge fresco (Heraeus Instruments GmbH)
  16. Nanodrop Spectrophotometer (VWR International, Peqlab)
  17. Mini-PROTEAN® Tetra Cell Casting Module (Bio-Rad Laboratories, catalog number: 1658008 )
  18. Buffer Tank and Lid (Bio-Rad Laboratories, catalog number: 1658040 )
  19. Mini Trans-Blot Module (Bio-Rad Laboratories, catalog number: 1703935 )
  20. Autoradiography casette
  21. FUSION SL-4 3500.WL (Chemiluminescence System) (PEQLAB)
  22. CCD camera FUSION 4 MP (VWR International, Peqlab)
  23. Orbital Shaker 3015 (GFL)
  24. Shaker (INFORS AG)
  25. Heating block (Gebr. Liebisch GmbH & Co. KG Labortechnik)

Software

  1. Fusion BIO-1D software

Procedure

Note: The preparation of buffers, solutions and SDS gels is explained in Recipes section.

  1. Recombinant protein overexpression and protein purification
    1. For overexpression of Aspergillus nidulans proteins in Escherichia coli (here: Rosetta DE3) inoculate 5 ml LB pre-culture, supplemented with appropriate antibiotics, for overnight incubation at 37 °C with agitation.
    2. The next morning, use 1 ml of the pre-culture to inoculate 1 L LB in a 2 L flask supplemented with appropriate antibiotics. Incubate the culture at 37 °C with ~220 rpm agitation for 2 to 3 h until OD600 of 0.6-0.7 is reached (depends on E. coli strain). OD600 can be determined with UV Spectrophotometer by taking 1 ml aliquot of the culture measured against a LB blank sample.
    3. When OD600 of 0.6-0.7 is reached, add 1 ml 1 M IPTG to the culture to induce the overexpression. Transfer the flask to room temperature or 16 °C for no longer than 20 h and 150-180 rpm agitation.
    4. Transfer the culture to 1 L centrifugation bottles, which should be pre-cooled on ice and then centrifuge at 4,000 rpm for 30 min at 4 °C.
    5. Discard the supernatant and keep the pellet on ice. Resuspend the pellet with 5-10 ml ice-cold affinity buffer.
    6. Transfer the resuspended pellet to a 50 ml tube and centrifuge at 4,000 rpm, 20 min, 4 °C.
    7. Discard the supernatant and store the pellet at -80 °C.
    8. For protein purification thaw the pellet on ice while preparing the lysis, affinity and elution buffers. Filter-degas the buffers using the filter and degassing system with MicronSep filters.
    9. Mix the pellet with 10 ml ice-cold lysis buffer per g pellet and resuspend by pipetting up and down (prevent air bubbles and keep solution on ice).
    10. Put the falcon in a box or glas with ice and sonicate the sample for approximately 8-10 rounds of 30 sec with 70% power and 1 min pause between each round. Take care that no air bubbles are formed during sonication and that the sample does not become warm.
    11. Transfer the lysed cells into SS34 centrifugation tubes and centrifuge at 20,000 rpm, 30 min, 4 °C.
    12. During centrifugation, wash the ÄKTA pumps and column with degassed 20% ethanol solution and degassed Millipore water and equilibrate with affinity (pump A) and elution (pump B) buffers.
    13. Transfer the supernatant to the superloop and connect the loop to the ÄKTA.
    14. Set the parameters for the ÄKTA run (pressue limit, flow, etc.) according to manufacturer recommendations. Start the run by setting the flow to 0.5 ml/min and press inject. The flow through can be collected. Switch from inject to load when the superloop is empty. Then wash with affinity buffer as long as UV reaches baseline again.
    15. Apply a gradient of 0- 50% elution buffer in 30 min and collect the flow through in 2 ml fractions. The protein of interest should elute during this gradient or when 100% elution buffer is applied to the column. Afterwards the ÄKTA system and column should be washed with degassed Millipore water and degassed 20% ethanol solution.
    16. The fractions containing the protein of interest can be analyzed by SDS PAGE. Therefore, take 20 µl samples and add 10 µl 3x loading dye, denature for 5 min at 95 °C and then load on a 12% SDS PAGE.
    17. Combine all fractions with proper amount of the protein of interest and concentrate them if necessary (e.g. to decrease the volume).
    18. Equilibrate the ÄKTA and size exclusion chromatography column in SEC buffer.
    19. Centrifuge the protein sample at 13,000 rpm for 10 min and inject the supernatant into the sample loop.
    20. Start the run with 0.35 ml/min flow and press inject. Collect the flow through and analyze peak fractions by SDS PAGE. Then combine the fractions which include the protein of interest, concentrate the solution and store at -20 °C.

  2. Preparation of protein crude extracts
    1. Take 100 ml autoclaved minimal media (MM) in 300 ml buffled falsks and add supplements according to the A. nidulans strains. Inoculate with 1 x 106 spores/ml medium and incubate at 37 °C for 20 hours under illumination and 120-150 rpm agitation.
    2. Collect the mycelium in funnels with miracloth filters. Wash the mycelium with 50-100 ml 0.96% NaCl solution and then take the miracloth filter containing the mycelium and dry it between paper towels.
    3. Transfer the dry mycelium (take mycel of the size of a 1 cent coin) to a 2 ml reaction tube and put into liquid nitrogen.
    4. Pre-cool the reaction tube holders of the Mixer Mill in liquid nitrogen by pouring a small amount into a Styrofoam box.
    5. Add two metal balls to each tube and put the tube into the holder.
    6. Close the holder and assemble them to the Mixer Mill. Set the program to 1 min and frequency to 30/sec then press start.
    7. Transfer the tube holder again into the box with a little bit of liquid nitrogen to prevent thawing.
    8. Take out the metal balls with a strong magnet. Then transfer the grinded mycelium to a new 2 ml tube and put the tubes again to liquid nitrogen.
    9. Put all tubes on ice and open them immediately. Add 300 µl B* buffer and vortex for ~1 min.
    10. Centrifuge for 30 min, 13,000 rpm, 4 °C.
    11. Transfer the supernatant to a fresh 1.5 ml tube and centrifuge again for 10 min, 13,000 rpm, 4 °C. In case there is still a pellet, transfer the supernatant to a fresh 1.5 ml tube.
    12. Measure the protein concentration with Nanodrop. To prevent protein aggregate formation, do not store the crude extracts longer than 1 week at -20 °C (to perform the deneddylation assay at the same or next day would be optimal).

  3. Deneddylation assay
    1. Protein crude extracts of csn deletion and wild type strains serve as neddylated and deneddylated cullin source. Recombinant A. nidulans CSN proteins are used to complement the deletions. If necessary, thaw all protein solutions, centrifuge at 13,000 rpm for 10 min at 4 °C (do not freeze and thaw protein solutions more than 2-3 times as proteins form aggregates). Transfer the supernatant to a new tube. Re-measure the concentration using Nanodrop.
    2. For each reaction, mix 80 µg of the protein crude extracts with 8 µg recombinant protein, 100 mM DTT and B* buffer to a final volume of 30 µl in a 1.5 ml reaction tube (Table 1). As a control, prepare the same reactions without recombinant protein. Incubate at 37 °C for 30 min and 120 rpm agitation.

      Table 1. Pipetting scheme (example)


    3. Add 15 µl 3x SDS sample buffer to each reaction, mix, denature at 95 °C for 5 min and then cool down on ice for 1 min. Flash spin the samples for 3-5 sec in a table centrifuge.
    4. Prepare 12% SDS gels.
    5. Load 10 µl of each sample on a gel and use 3.5 µl of Page Ruler Prestained Protein Ladder as size standard. Use 1x running buffer to run the electrophoresis. Apply 120 V until the proteins reach the resolving gel and then increase the voltage to 150-180 V until the 25 kDa band of the page ruler ran out of the gel.
    6. Stop the migration and carefully transfer the gel to the western blotting apparatus. Assemble the western blot according to the manufacturer recommendations and as represented in Figure 1. Close the cassette with the colorless site and transfer it into the Mini trans-blot central core after it was placed into the electrophoresis chamber filled with transfer buffer. The proteins can be blotted either for at least 1 h at 100 V and appropriate cooling of the buffer or overnight at 35 V.


      Figure 1. Western blot assembly. Schematic representation of the order of blot assembly for western hybridization.

    7. Disassemble the blot and cover the membrane with blocking solution for 1 h at room temperature and gentle agitation.
    8. Discard the blocking solution and separate the membrane into two pieces at the 70 kDa page ruler band (Figure 2). Add the primary antibody α-Nedd8 or α-CulA (dilution in blocking solution 1:1,000) to the upper membrane. As loading control the primary antibody α-Tub or α-Act (dilution in blocking solution 1:1,000) can be added to the lower membrane part. Incubate for 1 h at room temperature and gentle agitation.


      Figure 2. Western blot membrane. The dashed line indicates where the membrane needs to be cut in order to develop the loading control (Actin or Tubulin ~40 kDa) simultaneously with the CulA or Nedd8 antibody (signal ~100 kDa).

    9. Remove the primary antibodies and wash the membranes 3 x 10 min with 1x TBST at room temperature and gentle agitation.
    10. Add the secondary antibody α-goat rabbit to the upper membrane and α-goat mouse (both 1:1,000 in blocking solution) to the lower membrane for 1 h at room temperature and gentle agitation.
    11. Remove the secondary antibodies and wash the membranes 3 x 10 min with 1x TBST buffer at room temperature and gentle agitation.
    12. For signal detection prepare the detection solutions freshly. Then mix detection solution A with detection solution B on the membrane for 2 min at room temperature and gentle agitation. Subsequently place the membrane into a plastic foil and transport it via an autoradiography cassette to the Fusion imaging system (important: switch on the PC and Fusion imaging system before you are ready to use, because the camera needs approximately 10 min for cooling down).
    13. Click on ‘start preview’, place the membrane into the apparatus, click ‘Set-up’ → ‘Sensitivity’ → ‘Super Sensitivity’ → ‘Auto Exposure’ (takes 1-4 min to detect appropriate signals). Save the image as TIF file (also automatically saved as STB file).
    14. Analyze signals with Fusion BIO-1D software as followed:
      1. Click ‘Open an Image’ and then choose the image. Click ‘Optical Density’.
      2. A- Lane definition: click ‘New Lane’, a box with green lines appears (Figure 3). Drag around the signals to define the area to be analyzed. click ‘Select all’ → ‘Next >>’.
      3. B- Background subtraction: click ‘Do rolling ball’ → ‘Next >>’.
      4. C- Spot separation: click ‘Add separation’ or use given separations and place the brackets around band boundaries by dragging the cursor (Figure 4). Click ‘Next >>’.
      5. Untick ‘Concentration’, ‘Height’, Area’ and ‘Molecular Weight’ (only ‘Volume’ has a tick) (Figure 5). Press the ‘Excel’ button. An Excel window opens with the volume data representing the pixel intensity inside a defined boundary (Figure 6). Further quantify signals considering wild type signal and loading controls.


      Figure 3. BIO-1D, A-Lane definition. Screenshot of BIO-1D software showing a green lined box that can be dragged to define the analysis area.


      Figure 4. BIO-1D, C-Spot separation. Screenshot of BIO-1D software displaying the separation of the band boundaries by placing brackets.


      Figure 5. BIO-1D, Data export to Excel. Screenshot of BIO-1D software displaying the step where the value of the Volume has to be selected to be exported to Excel.


      Figure 6. BIO-1D, Data in Excel. Screenshot of Excel table with values of pixel intensity inside a defined boundary.

Representative Data

Figure 7 shows a typical result of an in vitro deneddylation assay similar to those shown in Beckmann et al. (2015). Usually most cullins are deneddylated in wild type. This is visible in the western hybridization and quantification of crude extracts from wild type (wt) where the Nedd8 antibody gives a weak signal at ~100 kDa, with CulA hybridization a strong signal for deneddylated CulA (CulA, **) and a slight signal for neddylated CulA (CulA-Nedd8, *) (Figure 7). In contrast, crude extracts of csn deletion strains show increased neddylated CulA amounts, which cannot be complemented by the addition of the respective recombinant CSN subunit to the deletion strain. The only exception where CulA is mostly deneddylated like in wild type is when recombinant CsnE is added to csnE deletion crude extract. Only then the CSN seems to be active.
Signals were quantified relative to the loading control and for Nedd8 relative to wild type. For CulA signals the ratio of neddylated CulA to non-neddylated CulA were calculated.


Figure 7. An example for in vitro deneddylation assay: CsnE can restore deneddylation activity of CSN. Western hybridization of A. nidulans crude extracts incubated with and without recombinant CSN proteins. A. The membrane was incubated with α-Nedd8 antibody resulting in a weak signal for wt and ΔcsnE + CsnE and strong signals for all other csn deletion strains with or without recombinant protein at ~100 kDa. α-Tub antibody was used as loading control. B. Detection with α-CulA underlined the result from (A) where the ratio of neddylated CulA (*) to deneddylated CulA (**) in ΔcsnE + recombinant CsnE was similar to the wild type and inverted in comparison to all other samples. Results were quantified using BIO1D software (Peqlab).

Notes

Users of this protocol should have experience in or collaborative groups for protein purification using the ÄKTA system.
For reliable data, at least three biological repetitions should be performed. This is also important for calculating error bars. Mean values and standard deviation were calculated using Excel with the formula below:

With σ as standard deviation, xi representing each value of the dataset, arithmetic mean value, n total number of data points, Σ the sum of (xi - x̄)2 of all data points.

Recipes

  1. Lysogeny broth (LB) medium
    1% (w/v) Peptone/Tryptone
    0.5% (w/v) yeast extract
    1% (w/v) NaCl
    Autoclave and supplement with antiobiotics (1:1,000) before usage.
  2. Lysis buffer
    500 mM NaCl
    50 mM Tris (pH 8.0)
    Add just before use: 1 mM PMSF
  3. Affinity buffer
    500 mM NaCl
    50 mM Tris (pH 8.0)
    5% (w/v) Imidazole in case of HisTrap columns
  4. Elution buffer for HisTrap
    500 NaCl
    50 mM Tris (pH 8.0)
    500 mM Imidazole
  5. Elution buffer for GSTrap
    500 NaCl
    50 mM Tris (pH 8.0)
    20 mM Glutathione
  6. Size exclusion buffer (SEC)
    120 mM NaCl
    20 mM Tris pH 8.0
    1 mM DTT
    1 mM EDTA
    5% (v/v) Glycerol
  7. Minimal medium
    1% (w/v) Glucose
    1x AspA
    2 mM MgSO4
    1x trace elements
  8. 50x AspA
    3.5 M NaNO3
    350 mM KCl
    560 mM KH2PO4
    Adjust to pH 5.5 with KOH (~30 mL 5 M KOH needed!), autoclave
  9. Trace elements
    18 µM FeSO4
    174 µM EDTA
    76 µM ZnSO4
    178 µM H3BO3
    25 µM MnCl2
    7.1 µM CoCl2
    6.4 µM CuSO4
    6.2 µM Na2MoO4
    Sterile filter
  10. B* buffer
    300 mM NaCl
    10 mM Tris-HCl (pH 7.5)
    0.5 mM EDTA
    0.2% (v/v) NP-40
    10% (v/v) Glycerol
    Add just before use:
    1 mM PMSF
    2 mM DTT
    5 mM E64
    100 µl/10 ml Complete Protease Inhibitor Cocktail
  11. 3x SDS sample buffer
    250 mM Tris-HCl (pH 6.8)
    15% 2-mercaptoethanol
    30% Glycerol
    7% SDS
    0.3% Bromophenol blue
  12. 12% SDS gels
    for 2 gels mix the following solutions in a 50 ml reaction tube:
    Resolving gel:
    5 ml 2x resolving buffer
    4 ml 30% acrylamide
    1 ml H2O
    150 µl 10% APS
    15 µl TEMED
    1. Invert the solution and then fill between the glas plates.
    2. Add ~1 ml 100% isopropanol on top of the gel to prevent air bubbles.
    3. Let the gels dry ~10-20 min.
    4. Then remove the isopropanol carefully.
    5. Use whatman paper to remove remaining drops.
    6. Then mix all components for the stacking gel in a 50 ml reaction tube.
    Stacking gel:
    2 ml 2x stacking buffer
    0.5 ml 30% acrylamide
    1.5 ml H2O
    60 µl 10% APS
    7.5 µl TEMED
    1. Invert the tube and then fill the stacking solution onto the resolving gel up to the top.
    2. Then place the comps carefully into the stacking gel.
    3. Let the gels dry for another 10-20 min.
  13. 2x resolving buffer
    0.75 M Tris-HCl (pH 8.8)
    0.1% (w/v) SDS
  14. 2x stacking buffer
    0.25 M Tris-HCl (pH 6.8)
    0.1% (w/v) SDS
  15. 10x SDS running buffer
    250 mM Tris-base
    2.5 M Glycine
    1% (w/v) SDS
    Dilute the buffer to 1x working solution with ddH2O
  16. 10 x transfer buffer
    250 mM Tris-base
    1.92 M glycine
    0.2% (w/v) SDS
    Dilute the buffer to 1x working solution with ddH2O
  17. 10x TBST (Tris-Buffered Saline Tween 20)
    100 mM Tris-HCl (pH 8.0)
    1.5 M NaCl
    0.5% (v/v) Tween-20
    Dilute the buffer to 1 x working solution with ddH2O
  18. Blocking solution
    5 % milk powder dissolved in 1x TBST buffer
  19. Detection solution A (prepare fresh just before use)
    9 ml ddH2O
    1 ml Tris-HCl (pH 8.5)
    100 µl 250 mM Luminol (dissolve stock solution in DMSO, protect from light, store at -20 °C)
    44 µl 400 mM p-coumaric acid (dissolve stock solution in DMSO, protect from light, store at -20 °C)
  20. Detection solution B (prepare fresh just before use)
    9 ml ddH2O
    1 ml 1 M Tris-HCl (pH 8.5)
    6.15 µl 30% H2O2
  21. Complete Protease Inhibitor Cocktail
    Dissolve 1 ½ tablets in 1 ml B*buffer

Acknowledgements

This protocol was adapted from a previously published study (Beckmann et al., 2015) and precursor experiments (Christmann et al., 2013; Harting et al., 2013; von Zeska Kress et al., 2012). We thank Dr. Elena Beckmann for her support and advice and Gabriele Heinrich for excellent technical assistance. We also thank Sabine Reen and Josua Schinke for careful proofreading the manuscript. This research has been supported by the Deutsche Forschungsgemeinschaft (DFG) within the SFB860.

References

  1. Beckmann, E. A., Köhler, A. M., Meister, C., Christmann, M., Draht, O. W., Rakebrandt, N., Valerius, O. and Braus, G. H. (2015). Integration of the catalytic subunit activates deneddylase activity in vivo as final step in fungal COP9 signalosome assembly. Mol Microbiol 97(1): 110-124.
  2. Christmann, M., Schmaler, T., Gordon, C., Huang, X., Bayram, Ö., Schinke, J., Stumpf, S., Dubiel, W. and Braus, G. H. (2013). Control of multicellular development by the physically interacting deneddylases DEN1/DenA and COP9 signalosome. PLoS Genet 9(2): e1003275.
  3. Harting, R., Bayram, Ö., Laubinger, K., Valerius, O. and Braus, G. H. (2013). Interplay of the fungal sumoylation network for control of multicellular development. Mol Microbiol 90(5): 1125-1145.
  4. von Zeska Kress, M. R., Harting, R., Bayram, Ö., Christmann, M., Irmer, H., Valerius, O., Schinke, J., Goldman, G. H. and Braus, G. H. (2012). The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development. Mol Microbiol 83(6): 1162-1177.

材料和试剂

  1. 重组蛋白过表达和蛋白质纯化
    1. 1ml比色杯10×10×45mm(Sarstedt AG,目录号:67.742)
    2. 色谱柱GSTrap(GE Healthcare,目录号:17-5130-01),HisTrap(GE Healthcare,目录号:17-5255-01),Superdex 200 HR 10/300GL(GE Healthcare,目录号:17-5175-01) ,HiPrep 16/60 Sephacryl S-300HR(GE Healthcare,目录号:17-1167-01)]
    3. Amicon Ultra离心过滤器(Merck Millipore Corporation)截留分子量(MWCO)取决于蛋白质大小[例如30kDa(Merck Millipore Corporation,MWCO,目录号:UFC903024)]
    4. MicronSep滤纸,0.22,47mm(Carl Roth GmbH + Co.,目录号:A009.1)
    5. 大肠杆菌(大肠杆菌)菌株 用过表达转化的过表达(例如,Rosetta DE3) 质粒包括感兴趣的具有N-或C-末端的蛋白质序列 谷胱甘肽-S-转移酶(GST-)或组氨酸(His-)标签
    6. 用作1%溶液的来自酪蛋白(Carl Roth GmbH + Co.,目录号:8952.2)的胰蛋白胨/蛋白胨
    7. 用作0.5%溶液的酵母提取物(Oxoid,目录号:LP0021)
    8. 用作1%溶液的氯化钠(NaCl)(Carl Roth GmbH + Co.,目录号:9265.2)
    9. 根据E的抗生素。大肠杆菌菌株
    10. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Carl Roth GmbH + Co., 目录号:2316.4)用作1M溶液,过滤灭菌
    11. 99.5%变性乙醇和1%MEK(VWR International,目录号: 85033.460),用作在ddH 2 O中稀释的20%溶液,过滤器脱气
    12. 咪唑
    13. 谷胱甘肽
    14. 溶菌酶培养基(LB)培养基(参见配方)
    15. 裂解缓冲液(见配方)
    16. 亲和缓冲区(请参阅配方)
    17. HisTrap的洗脱缓冲液(参见配方)
    18. GSTrap的洗脱缓冲区(参见配方)
    19. 尺寸排阻缓冲液(SEC)(参见配方)

  2. 制备蛋白粗提物
    1. 纸毛巾
    2. Aspergillus nidulans 菌株的孢子
    3. Miracloth(Merck Millipore Corporation,目录号:475855-1R)
    4. 用作0.95%溶液的氯化钠(NaCl)(Carl Roth GmbH + Co.,目录号:9265.2)
    5. 液氮(空气液体)
    6. 最小培养基(见配方)
    7. 50x AspA(请参阅食谱)
    8. 微量元素(参见配方)
    9. B *缓冲区(参见配方)

  3. 丁二烯基化测定
    1. 凝胶吸印纸(Schleicher& Schuell BioScience GmbH,目录号:10426694)
    2. 印迹膜Amersham TM Protran TM sup0.45μmNC(GE Healthcare,目录号:10600002)
    3. 甲醇(VWR International,目录号:20864.320)
    4. 2-丙醇HiPerSolv CHROMANORM(VWR International,目录号:20880.320)
    5. 页尺寸预染蛋白梯(Thermo Fisher Scientific,目录号:26616)
    6. 丙烯酰胺Rotiphorese Gel 30(37,5:1)(Carl Roth GmbH + Co.,目录号:3029.1)
    7. Sucofin脱脂奶粉(TSI GmbH,目录号:ST00227)
    8. α-CulA,α-Nedd8抗体(GenScript)
    9. 单克隆α-微管蛋白抗体(Sigma-Aldrich,目录号:T0926)
    10. α-肌动蛋白抗体(Novus Biologicals,目录号:NB100-74340)
    11. α-山羊兔二抗(Thermo Fisher Scientific,Invitrogen TM ,目录号:G21234)
    12. α-山羊小鼠二抗(Jackson ImmunoResearch,目录号:115-035-003)
    13. 对 - 香豆酸(Sigma-Aldrich,目录号:C9008-5G) 将400mM储备溶液溶解于DMSO中(避光保存 -20°C)
    14. 鲁米诺(Carl Roth GmbH + Co.,目录号:4203.1) 用作溶于DMSO的250mM储备溶液(避光, 存储在-20°C)
    15. 2-巯基乙醇(Sigma-Aldrich,Fluka Chemika,目录号:63700)
    16. 用作7%,0.1%,1%,0.2%溶液的十二烷基硫酸钠(SDS)(Carl Roth GmbH + Co.,目录号:CN30.3)
    17. 用作0.3%溶液的溴酚蓝(Sigma-Aldrich,Riedel-deHa?n,目录号:32768)
    18. 使用10%溶液的过硫酸铵(APS)(Sigma-Aldrich,Fluka Analytical,目录号:09913-100G)
    19. 四甲基乙二胺(TEMED)(Carl Roth GmbH + Co.,目录号:2367.1)
    20. Tween-20(Carl Roth GmbH + Co.,目录号:9127.1)
    21. 用作250mM溶液的Tris-PUFFERAN (Carl Roth GmbH + Co.,目录号:4855.3)
    22. 用作2.5M溶液的大豆(Carl Roth GmbH + Co.,目录号:3187.3)
    23. 30%过氧化氢(H 2 O 2)(Merck Millipore Corporation,目录号:822287.2500)
    24. 3x SDS样品缓冲液(见配方)
    25. 12%SDS凝胶(见配方)
    26. 2x分辨缓冲液(参见配方)
    27. 2x堆叠缓冲区(参见配方)
    28. 10x SDS运行缓冲液(参见配方)
    29. 10x传输缓冲区(参见配方)
    30. 10x TBST(Tris-缓冲盐水Tween 20)缓冲液(参见配方)
    31. 阻止解决方案(参见配方)
    32. 检测溶液A(参见配方)
    33. 检测溶液B(参见配方)

  4. 其他(用于多种方法的材料和试剂)
    1. 微管1.5ml(Sarstedt AG,目录号:72.690.001)
    2. 微管2.0ml(Sarstedt AG,目录号:72.691)
    3. 管115×28mm,PP(Sarstedt AG,目录号:62.559.001)
    4. 5ml试管
    5. 2升烧瓶
    6. PPCO离心瓶(Slaughte,Nalgene,目录号:525-2316)
    7. Oak-Ridge离心管,50ml(Carl Roth GmbH + Co.,目录号:PK03.1)
    8. 300ml带挡板的烧瓶中
    9. 渠道
    10. 碎冰
    11. 用作1M溶液的1,4-二硫苏糖醇(DTT)(Carl Roth GmbH + Co.,目录号:6908.2)
    12. 反式琥珀酰-L-亮氨酰氨基(4-胍基)丁烷,E-64蛋白酶 ?抑制剂(Sigma-Aldrich,目录号:E3132) 解决方案
    13. 用作100mM异丙醇溶液的苯甲基磺酰氟(PMSF)(Carl Roth GmbH + Co.,目录号:6367.3)
    14. 双蒸馏水(ddH 2 O)
    15. 微孔水
    16. 用作2M溶液的氯化钠(NaCl)(Carl Roth GmbH + Co.,目录号:9265.2)
    17. 三盐酸盐(Tris-HCl)(Carl Roth GmbH + Co.,目录 number:9090.3)
      注意:准备一系列具有不同浓度和pH的Tris-HCl溶液,包括pH 6.8,7.5,8.0和pH 8.8的1M溶液;具有pH 8.8的0.75M溶液;具有pH 8.0的100mM溶液。
    18. 用作0.5mM溶液的乙二胺四乙酸(EDTA)(Carl Roth GmbH + Co.)
    19. Nonoxinol-40(NP-40)(Affymetrix,USB Corporation,目录号:19628)
    20. 甘油(Carl Roth GmbH + Co.,目录号:3783.1)
    21. 完全蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11873580001)(参见Recipes)

设备

  1. Biowave DNA Llife Science分光光度计(Biochrom WPA,目录号:80-3004-70)
  2. 离心Sorvall RC3B Plus(Thermo Fisher Scientific)
  3. 离心Sorvall RC5B Plus(Thermo Fisher Scientific)
  4. Rotor Sorvall SS34(Thermo Fisher Scientific)
  5. ?KTAexplorer10(GE Healthcare,目录号:18-1300-00)
  6. pH计pH526(Sigma-Aldrich,型号:WTW)
  7. 过滤器和脱气系统(Satorius)
  8. 真空泵(KNF NEUBERGER,目录号:2.03360238)
  9. 超声波Sonoplus HD2070(Bandelin)
  10. Superloop 1/16"(GE Healthcare,目录号:18-1113-82)
  11. 样品环5ml(GE Healthcare,目录号:18-1140-53)
  12. 搅拌机MM 400(Retsch)
  13. Vortex Genie 2(科学工业)
  14. 移液管(1-5,000μl)(Gilson)
  15. 离心机Biofuge fresco(Heraeus Instruments GmbH)
  16. Nanodrop分光光度计(VWR International,Peqlab)
  17. Mini-PROTEAN Tetra Cell Casting Module(Bio-Rad Laboratories,目录号:1658008)
  18. 缓冲罐和盖(Bio-Rad Laboratories,目录号:1658040)
  19. Mini Trans-Blot Module(Bio-Rad Laboratories,目录号:1703935)
  20. 自动射线照相机
  21. FUSION SL-4 3500.WL(化学发光系统)(PEQLAB)
  22. CCD相机FUSION 4MP(VWR International,Peqlab)
  23. 轨道振动器3015(GFL)
  24. Shaker(INFORS AG)
  25. 加热块(Gebr.Leebisch GmbH& Co.KG Labortechnik)

软件

  1. Fusion BIO-1D软件

程序

注意:"配方"部分介绍了缓冲液,溶液和SDS凝胶的制备

  1. 重组蛋白过表达和蛋白质纯化
    1. 对于 大肠杆菌中的构巢曲霉(Aspergillus nidulans)蛋白的表达(这里:Rosetta DE3)接种5ml LB预培养物,补充有 适当的抗生素,在37℃下孵育过夜 搅动。
    2. 第二天早上,使用1毫升的预培养 在补充适当的2L烧瓶中接种1L LB 抗生素。孵育培养物在37℃,?220 rpm搅拌2 ?到3小时,直到OD 600达到0.6-0.7(取决于大肠杆菌菌株)。 OD 600可以用UV分光光度计通过取1ml等分试样来测定 ?的针对LB空白样品测量的培养物
    3. 当OD 600 时 ?0.6-0.7,向培养物中加入1ml 1M IPTG以诱导 过表达。将烧瓶转移至室温或16℃,没有 超过20小时和150-180rpm搅拌
    4. 转移文化 至1L离心瓶,其应在冰上预冷却 然后在4℃下以4,000rpm离心30分钟。
    5. 弃去上清液,保持沉淀在冰上。用5-10ml冰冷的亲和缓冲液重悬沉淀
    6. 将重悬的颗粒转移到50ml管中,并在4,000rpm,20分钟,4℃离心。
    7. 弃去上清液,将沉淀储存于-80℃
    8. 对于蛋白质纯化,在冰上融化沉淀物 裂解,亲和和洗脱缓冲液。过滤器使用缓冲器 过滤器和除气系统与MicronSep过滤器
    9. 混合 用每克沉淀物10ml冰冷的裂解缓冲液沉淀,并重悬 上下移动(防止气泡并将溶液保持在冰上)
    10. 把猎鹰放在一个盒子或格拉斯冰和超声的样品 持续约8-10轮30秒,70%功率和1分钟暂停 每轮之间。注意,在此期间不会形成气泡 超声处理和样品不变暖。
    11. 将裂解的细胞转移到SS34离心管中,并在20,000 rpm,30分钟,4℃离心
    12. 在离心过程中,用脱气的方法清洗?KTA泵和柱 ?20%乙醇溶液和脱气的Millipore水中并平衡 亲和力(泵A)和洗脱(泵B)缓冲液。
    13. 将上清液转移到超级环,将环连接到?KTA。
    14. 设置?KTA运行的参数(按下限制,流量,等) 根据制造商的建议。通过设置启动运行 流速至0.5ml/min并按压注射。可以收集流过。 当超级环路为空时,从注入切换到加载。然后用 亲和缓冲液,只要UV再次达到基线
    15. 应用a 在30分钟内梯度为0-50%洗脱缓冲液并收集流过 ?以2ml级分。感兴趣的蛋白质在此期间应洗脱 梯度或当100%洗脱缓冲液应用于柱时。 然后?KTA系统和色谱柱应用脱气洗涤 Millipore水和脱气的20%乙醇溶液
    16. 馏分 ?可以通过SDS PAGE分析含有目的蛋白质的蛋白质。 因此,取20μl样品,加入10μl3x负载染料,变性 ?在95℃下5分钟,然后在12%SDS PAGE上加样
    17. 结合所有 适当量的目标蛋白质和浓缩物的级分 如果需要(例如减少音量)。
    18. 在SEC缓冲液中平衡?KTA和尺寸排阻色谱柱。
    19. 将蛋白质样品在13,000 rpm离心10分钟,并将上清液注入样品环
    20. 以0.35ml/min流速开始运行并按压注射。收集 流过并通过SDS PAGE分析峰级分。然后结合 包括目标蛋白质的级分,浓缩 溶液并储存在-20°C。

  2. 制备蛋白粗提物
    1. 取100毫升高压灭菌的基本培养基(MM)在300毫升的buffled falsks 并根据 A添加补充。构巢曲霉菌株。接种 ?1×10 6孢子/ml培养基并在37℃下孵育20小时 照明和120-150rpm搅拌
    2. 收集菌丝体 漏斗与miracloth过滤器。用50-100ml 0.96% NaCl溶液,然后取含有菌丝体的miracloth过滤器 ?并在纸巾之间擦干
    3. 转移干菌丝体(取1厘米硬币大小的mycel)到2毫升反应管,并放入液氮。
    4. 在液氮中预冷却搅拌机的反应管支架,少量注入聚苯乙烯泡沫塑料盒中。
    5. 在每个管子上加两个金属球,将管子放入支架中
    6. 关闭夹具并将它们组装到搅拌机上。将程序设置为1分钟,频率设置为30 /秒,然后按开始。
    7. 用一点液氮将管夹再次转移到箱子中,以防止解冻。
    8. 取出带有强力磁铁的金属球。然后转移 研磨菌丝体至新的2ml管,并将管再次置于液体中 氮。
    9. 将所有管放在冰上,立即打开他们。加入300μlB *缓冲液并涡旋约1分钟
    10. 离心30分钟,13,000 rpm,4℃
    11. 转移上清液到新鲜的1.5ml管,离心 再次10分钟,13,000rpm,4℃。如果仍有颗粒, 将上清液转移至新鲜的1.5ml管中。
    12. 测量 蛋白质浓度与Nanodrop。防止蛋白聚集 形成,不存储长于1周的粗提取物在-20℃ (在相同或次日进行deneddylation测定) 最佳)。

  3. 丁二烯基化测定
    1. 蛋白质粗提取物 的csn 缺失和野生型菌株作为neddylated和 去甲二烯基化的卡蛋白源。重组构巢曲霉 CSN蛋白是 用于补充缺失。如果需要,解冻所有蛋白质 溶液,在4℃下以13,000rpm离心10分钟(不冻结 并融化蛋白质溶液超过2-3倍的蛋白质形式 聚集体)。转移上清液到一个新的管。重新测量 使用Nanodrop浓度
    2. 对于每个反应,混合80μg的 ?蛋白质粗提物与8μg重组蛋白,100mM DTT和B * ?缓冲液至1.5ml反应管中的最终体积为30μl(表1)。 作为对照,制备没有重组蛋白的相同反应。 在37℃孵育30分钟和120rpm搅拌。

      表1.移液方案(示例)


    3. 向每个反应中加入15μl3x SDS样品缓冲液,混合,在95℃变性 ?5分钟,然后在冰上冷却1分钟。闪光旋转 样品在台式离心机中3-5秒
    4. 准备12%SDS凝胶
    5. 加载10微升的每个样品在凝胶上,并使用3.5微升的页面尺 预染蛋白梯度作为大小标准。使用1x运行缓冲区运行 ?电泳。施加120 V,直到蛋白质达到解决 ?凝胶,然后将电压升至150-180 V,直到25 kDa谱带 ?页面标尺跑出凝胶。
    6. 停止迁移和 小心地将凝胶转移到western印迹装置。组装 根据制造商的建议和as 如图1所示。关闭带有无色位点的盒式磁带 在放置后将其转移到小转染印迹中心核中 进入填充有转移缓冲液的电泳室。的 蛋白质可以在100V和适当时印迹至少1小时 ?冷却缓冲液或在35V过夜。


      图1.蛋白质印迹装配。用于西方杂交的印迹装配顺序的示意图
    7. 拆开印迹并用封闭溶液在室温下温育1小时并轻轻摇动覆盖膜
    8. 弃去封闭溶液,将膜分成两部分 片段在70 kDa页尺子带(图2)。添加主要 抗体α-Nedd8或α-CulA(在封闭溶液中稀释1:1,000)至 上膜。作为加载控制初级抗体α-Tub或 α-Act(在封闭溶液中稀释1:1,000)可以加入到下层 膜部分。在室温下孵育1小时,温和 搅拌

      图2.蛋白质印迹膜。虚线 指示膜需要被切割以便发展 加载对照(肌动蛋白或微管蛋白?40kDa)与CulA同时 或Nedd8抗体(信号?100kDa)
    9. 取出一抗,并在室温下用1x TBST洗涤膜3×10分钟,轻轻搅拌
    10. 加入第二抗体α山羊兔上部膜和 α-山羊小鼠(两者在封闭溶液中为1:1,000)至下膜 在室温下温和搅拌1小时
    11. 去除 次级抗体,并用1x TBST洗涤膜3×10分钟 缓冲液在室温下轻轻搅拌
    12. 对于信号 检测新鲜制备检测溶液。然后混合检测 溶液A与检测溶液B在膜上室温2分钟 温度和温和搅拌。随后将膜放入 ?塑料箔,并通过放射自显影盒运输 融合成像系统(重要:打开PC和融合成像 系统之前就准备好使用,因为相机需要 约10分钟用于冷却)。
    13. 点击"开始" 预览',将膜放入设备,单击'设置'→ '敏感度'→'超级敏感度'→'自动曝光'(需1-4分钟 检测适当的信号)。将图像另存为TIF文件(也 自动保存为STB文件)。
    14. 使用Fusion BIO-1D软件分析信号如下:
      1. 点击"打开图片",然后选择图片。点击"光密度"
      2. A-频道定义:点击"新频道 ?(图3)。在信号周围拖动以定义要分析的区域。 点击"全部选择""→"下一步>>'。
      3. B-背景扣除:点击"滚动球"→"下一步">>'。
      4. C-点分隔:点击"添加分隔"或使用指定的分隔 并通过拖动光标将括号放在带边界附近 (图4)。点击"下一步">>'。
      5. 取消'浓度', "(只有" "具有刻度),"高度","区域 (图5)。按" Excel "按钮。将打开一个Excel窗口 表示限定边界内的像素强度的体数据 (图6)。考虑野生型信号和进一步量化信号 加载控件。


      图3. BIO-1D,A-Lane定义。屏幕截图 ?BIO-1D软件显示一个绿色的内衬盒,可以拖动来定义 ?分析区。


      图4. BIO-1D,C-spot分离。屏幕截图 的BIO-1D软件显示带边界的分离 放置括号

      图5. BIO-1D,数据导出到Excel ?的BIO-1D软件显示的步骤,其中Volume的值 必须选择导出到Excel

      图6. BIO-1D,Excel中的数据。Excel表格中包含已定义边界内像素亮度值的屏幕截图。

代表数据

图7显示了类似于Beckmann等人(2015)中所示的体外脱离子化测定的典型结果。通常大多数cullins在野生型中是deneddylated。这在来自野生型(wt)的粗提取物的western杂交和定量中是可见的,其中Nedd8抗体在?100kDa处给出弱信号,CulA杂交对于去甲二基化CulA(CulA,**)具有强信号,对于Nedd化的CulA(CulA-Nedd8,*)(图7)。相比之下,csn 缺失菌株的粗提取物显示增加的Nedd化的CulA量,其不能通过向缺失菌株中添加相应的重组CSN亚基来补充。唯一的例外是其中CulA主要是deneddylated,如在野生型中,当重组 cnE 被添加到csnE缺失粗提取物时。只有这样,CSN似乎是活跃的。
相对于装载对照和Nedd8相对于野生型来定量信号。对于CulA信号,计算Nedd化的CulA与非Nedd化的CulA的比例。


图7. 体外 deneddylation测定的实例:CsnE可以恢复CSN的deneddylation活性。用非重组CSN蛋白孵育的构巢曲霉粗提物。 A.将膜与α-Nedd8抗体一起孵育,导致对于wt和ΔcsnE+ CsnE的弱信号,对于具有或不具有?100kDa的重组蛋白的所有其他csn 缺失菌株的强信号。使用α-Tub抗体作为加样对照。 B.用α-CulA检测下划线的是来自(A)的结果,其中在ΔcysE +重组CsnE中的Neddylated CulA(*)与deneddylated CulA(**)的比例与野生型并与所有其他样品相比反转。使用BIO1D软件(Peqlab)定量结果。

笔记

该协议的用户应该有使用?KTA系统进行蛋白质纯化的经验或协作组。
对于可靠的数据,应进行至少三次生物重复。这对计算误差条也很重要。使用具有以下公式的Excel计算平均值和标准偏差:

以表示数据集的每个值的σ作为标准偏差, x i >算术平均值, n 总数据点,Σ -

食谱

  1. 溶菌酶培养基(LB)培养基
    1%(w/v)蛋白胨/胰蛋白胨 0.5%(w/v)酵母提取物
    1%(w/v)NaCl 高压灭菌并在使用前补充抗生素(1:1,000)
  2. 裂解缓冲液
    500 mM NaCl
    50mM Tris(pH8.0) 使用前加入:1mM PMSF
  3. 亲和缓冲区
    500 mM NaCl
    50mM Tris(pH8.0) 在HisTrap柱
    的情况下为5%(w/v)咪唑
  4. HisTrap的洗脱缓冲区
    500 NaCl
    50mM Tris(pH8.0) 500mM咪唑
  5. GSTrap的洗脱缓冲区
    500 NaCl
    50mM Tris(pH8.0) 20mM谷胱甘肽
  6. 大小排除缓冲区(SEC)
    120 mM NaCl 20mM Tris pH 8.0 1 mM DTT
    1mM EDTA
    5%(v/v)甘油
  7. 最小培养基
    1%(w/v)葡萄糖 1x AspA
    2mM MgSO 4 1个微量元素
  8. 50x AspA
    3.5 M NaNO 3 3/
    350 mM KCl
    560mM KH 2 PO 4 sub/
    用KOH(?30mL 5M KOH需要!),高压釜
    调节到pH 5.5
  9. 微量元素
    18μMFeSO 4
    174μMEDTA
    76μMZnSO 4
    178μMH sub 3 BO 3
    25μMMnCl 2
    7.1μMCoCl 2
    6.4μMCuSO 4
    6.2μMNa 2 MoO 4 sub / 无菌过滤器
  10. B *缓冲区
    300 mM NaCl
    10mM Tris-HCl(pH7.5) 0.5mM EDTA 0.2%(v/v)NP-40
    10%(v/v)甘油 在使用前添加:
    1mM PMSF
    2 mM DTT
    5 mM E64
    100μl/10ml完全蛋白酶抑制剂混合物
  11. 3x SDS样品缓冲液
    250mM Tris-HCl(pH6.8)
    15%2-巯基乙醇 30%甘油
    7%SDS
    0.3%溴酚蓝
  12. 12%SDS凝胶
    对于2种凝胶,将以下溶液混合在50ml反应管中:
    解决凝胶:
    5 ml 2x分离缓冲液
    4ml 30%丙烯酰胺 1ml H 2 O 2 / 150μl10%APS
    15μlTEMED
    1. 倒置溶液,然后在玻璃板之间填充。
    2. 在凝胶顶部加入约1ml 100%异丙醇以防止气泡。
    3. 让凝胶干燥?10-20分钟。
    4. 然后小心地除去异丙醇。
    5. 使用whatman纸去除剩余的液滴。
    6. 然后将所有用于堆积凝胶的组分混合在50ml反应管中
    堆叠胶:
    2ml 2x堆积缓冲液
    0.5ml 30%丙烯酰胺 1.5ml H 2 O 2 / 60微升10%APS
    7.5μlTEMED
    1. 倒置管,然后将堆叠溶液填充到溶解凝胶上至顶部。
    2. 然后将comps小心地放入堆叠凝胶。
    3. 让凝胶干燥10-20分钟。
  13. 2x解析缓冲区
    0.75M Tris-HCl(pH8.8)
    0.1%(w/v)SDS
  14. 2x堆叠缓冲区
    0.25M Tris-HCl(pH 6.8)
    0.1%(w/v)SDS
  15. 10x SDS运行缓冲液
    250 mM Tris-base
    2.5 M甘氨酸 1%(w/v)SDS
    使用ddH 2 O缓冲液稀释至1x工作溶液
  16. 10 x转移缓冲区
    250 mM Tris-base
    1.92M甘氨酸 0.2%(w/v)SDS
    使用ddH 2 O缓冲液稀释至1x工作溶液
  17. 10x TBST(Tris-缓冲盐水Tween 20)
    100mM Tris-HCl(pH8.0) 1.5 M NaCl
    0.5%(v/v)Tween-20 用ddH 2 O缓冲液稀释至1×工作溶液
  18. 封锁解决方案
    5%奶粉溶于1x TBST缓冲液中
  19. 检测溶液A(使用前准备新鲜)
    9ml ddH 2 O 2 / 1ml Tris-HCl(pH8.5) 100μl250mM鲁米诺(溶于DMSO的储备溶液,避光,-20℃保存) 44μl400 mM对香豆酸(溶于DMSO中的储备溶液,防光,-20°C储存)
  20. 检测溶液B(在使用前准备新鲜)
    9ml ddH 2 O 2 / 1ml 1M Tris-HCl(pH8.5) 6.15μl30%H 2 O 2 sub。
  21. 完全蛋白酶抑制剂鸡尾酒
    将1?片溶于1ml B *缓冲液中

致谢

该方案改编自先前发表的研究(Beckmann等人,2015)和前体实验(Christmann等人,2013; Harting等人,/em>,2013; von Zeska Kress et al。,2012)。我们感谢Elena Beckmann博士的支持和建议,以及Gabriele Heinrich的优秀技术援助。我们也感谢Sabine Reen和Josua Schinke仔细校对手稿。这项研究得到了在SFB860内的德国Forschungsgemeinschaft(DFG)的支持。

参考文献

  1. Beckmann,E.A.,K?hler,A.M.,Meister,C.,Christmann,M.,Draht,O.W.,Rakebrandt,N.,Valerius,O.and Braus,G.H。 催化亚基的整合在体内激活deneddylase活性作为最后一步真菌COP9信号核糖体装配。 Mol Microbiol 97(1):110-124。
  2. Christmann,M.,Schmaler,T.,Gordon,C.,Huang,X.,Bayram,?。,Schinke,J.,Stumpf,S.,Dubiel,W.and Braus,G.H。 通过物理相互作用的deneddylases DEN1/DenA和COP9信号体控制多细胞发育。 em> PLoS Genet 9(2):e1003275。
  3. Harting,R.,Bayram,?。,Laubinger,K.,Valerius,O.和Braus,G.H。(2013)。 真菌s??umoylation网络的相互作用,用于控制多细胞发育。 Mol Microbiol 90(5):1125-1145。
  4. von Zeska Kress,M.R.,Harting,R.,Bayram,?。,Christmann,M.,Irmer,H.,Valerius,O.,Schinke,J.,Goldman,G.H.and Braus,G.H。(2012)。 COP9信号体抵抗真菌发育过程中cullin SCF泛素E3 RING连接酶的积累。 Mol Microbiol 83(6):1162-1177。
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。

X


How to cite this protocol: Köhler, A. M., Meister, C. and Braus, G. H. (2016). In vitro Deneddylation Assay. Bio-protocol 6(6): e1756. DOI: 10.21769/BioProtoc.1756; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)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。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook