CN105445472A - Method and kit for detecting activity of micro-molecular G protein Rap1 - Google Patents
Method and kit for detecting activity of micro-molecular G protein Rap1 Download PDFInfo
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- CN105445472A CN105445472A CN201510776243.9A CN201510776243A CN105445472A CN 105445472 A CN105445472 A CN 105445472A CN 201510776243 A CN201510776243 A CN 201510776243A CN 105445472 A CN105445472 A CN 105445472A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention belongs to the field of molecular biology and discloses a method and kit for detecting the activity of a micro-molecular G protein Rap1 by using GST pull-down and Western-Blot methods. The kit comprises a conventional GST pull-down experiment assembly, a GST-Ra1GDS-RBD fused protein which is specifically combined with Rap1-GTP, and an antibody for specifically identifying the Rap1. When the method and kit for detecting the activity of the micro-molecular G protein Rap1 are used for detecting the activity of the Rap1, the method and the kit have the advantages of rapidness, accuracy and simple method, and are beneficial to clinical and research utilization.
Description
Technical field
The invention belongs to molecular biological arts, a kind of method and kit detecting small molecular G protein Rap1 activity.
Background technology
Small molecular G protein Rap1 belongs to Ras family, and its similar, in Ras, is in activated state (Rap1-GTP) after in conjunction with GTP, in conjunction with being then in inactive state (Rap1-GDP) after GDP.In cell, Rap1 plays molecular switch by the dynamic translation between Rap1-GTP and Rap1-GDP, and extracellular signal is combined by specific guanine nucleotide acid exchange factor regulation and control Rap1 and GTP, thus activates Rap1; Born of the same parents' internal specific gtpase activating protein promotes GTP hydrolysis, thus makes Rap1 inactivation.The Rap1 signal of activation regulates and controls different biological functions by the signaling molecule that its downstream is different.Born of the same parents' external stimulus factor is growth factor, cell factor, chemotactic factor (CF) and second messenger Ca such as
2+, DAG, cAMP etc., activate Rap1 thus regulate and control different signal paths by guanine nucleotide exchange factor; And the Cannabined receptor of activation can make the Rap1 signal be activated be in state of activation all the time by lowering gtpase activating protein.Therefore, the physiological function of Rap1 signal mediation presents diversity, and on cell proliferation, differentiation, adhesion, migration etc. play different regulating and controlling effects.Rap1 passes through Raf/ERK
1/2, p38/MAPK/CREB or PI3K modulate tumor cell propagation, also affect infiltration and the migration of tumour cell by regulating the cell adhesion of cadherin mediation; In lymphatic system, the pathologic sustained activation of Rap1 causes myeloid leukemia; In nervous system, Rap1 signal can promote neuron polarity formation and axon growth, can also regulate neurite outgrowth.Rap1 signal can regulate and control the Changes of Plasticity of nerve synapse 26S Proteasome Structure and Function, also has correlativity with neuronic migration.Therefore, detect the activity of Rap1 albumen for propagation and the migration of understanding tumour, and it is significant to study its function in nervous system.
Summary of the invention
One of goal of the invention of the present invention is to be to provide a kind of method detecting small molecular G protein Rap1 activity, utilizes the method can detect the activity level of Rap1 in the clone of tissue or in vitro culture.
Two of object of the present invention be to utilize said method detect small molecular G protein Rap1 activity kit.
For solving the problem, the invention provides comprise glutathione sepharose beads, IPbuffer, SDS-PAGEloadingbuffer, protease inhibitors, inhibitors of phosphatases and can specific binding Rap1-GTP GST-RalGDS-RBD fusion with can the kit of specific recognition Rap1 antibody and detection method.
Its technical solution is as follows: a kind of method detecting small molecular G protein Rap1 activity, can the characteristic of specific bond Rap1-GTP according to the Rap1 binding structural domain (RBD) of hRalGDS gene, build GST-RalGDS-RBD prokaryotic expression plasmid, obtain GST-RalGDS-RBD albumen through induction purifying, and utilize GSTpull-down and Western-Blot method to detect the method for small molecular G protein Rap1 activity;
Its concrete steps are as follows:
Step 1 builds GST-RalGDS-RBD prokaryotic expression plasmid
RalGDS is the effector in Rap1 downstream, containing Rap1 binding structural domain (being positioned at its 798-885 amino acids residue), and can directly in conjunction with Rap1-GTP.In inquiry ncbi database, hRalGDS gene pairs answers the coded sequence of 798-885 amino acids residue, the multiple clone site (shown in Fig. 1) of contrast prokaryotic expression carrier pGEX-6P-1, BamHI and EcoRI site is wherein selected to design upstream primer (5 '-CGCGGATCCGCGctgccgctctacaacc-3 ') and downstream primer (5 '-CCGGAATTCCGGtcagaagatgcccttggcaa-3 ') as restriction enzyme site, with people liver library for masterplate, pcr amplification obtains object fragment.After agarose gel electrophoresis is separated, reclaim kit with gel reclaim, the object fragment reclaimed and pGEX-6P-1 empty carrier are respectively after BamHI and EcoRI double digestion, again through agarose gel electrophoresis separation and recovery, the object fragment of recovery and carrier mix with the ratio of 5:1.Then T is used
4dNA ligase spends the night in 16 DEG C of connections, transforms DH5 α Escherichia coli subsequently, be coated with flat board and the positive colony selected wherein by this system, and selected positive colony obtains the object fragment (shown in Fig. 2) of about 418bp after restriction enzyme is cut.The coded sequence that GST-RalGDS-RBD prokaryotic expression plasmid checks order in the object fragment and ncbi database that obtain completely the same (shown in Fig. 3).This shows that GST-RalGDS-RBD prokaryotic expression plasmid successfully constructs.
The prokaryotic expression of step 2GST-RalGDS-RBD fusion and purifying
With the GST-RalGDS-RBD Plastid transformation BL21 Escherichia coli of above-mentioned structure, transfer in 100mLLB nutrient culture media by 1%, 37 DEG C of 180r/min shaken cultivation are to exponential phase (D
600nm=0.6); IPTG is added subsequently, 20 DEG C of 180r/min vibration overnight induction by 100 μMs/L final concentration; Next day 4 DEG C of 4000rpm10min collected by centrifugation bacterium liquid, by resuspended for the Escherichia coli 30mL damping fluid PB [containing 0.5mM/LDTT, 2 μ g/mLAprotimin, 2 μ g/mLLeupeptin, 1mM/LPMSF, 1mM/LNa3VO4,10mM/LNaF in 30mLPBS] obtained, then high pressure is broken, and ultrasound wave is broken further subsequently.4 DEG C of centrifugal pyrolysis products of 13000rpm10min, get supernatant, the supernatant that the 300 μ L glutathione sepharose beads in advance through overbalance obtain after centrifugal is mixed, 4 DEG C of DLs turn 2h, then the PB buffer solution glutathione sepharose beads of precooling, utilize elution buffer [5ml fills a prescription: 250 μ L1M/LTris-Cl, ph7.6,4.75mL ultrapure water, 0.0154g reduced glutathione] that GST-RalGDS-RBD is washed from gel beads.SDS-PAGE coomassie brilliant blue staining method is finally utilized to detect the GST-RalGDS-RBD fusion (shown in Fig. 4, size is about 40kDa) of purifying.
Step 3 uses GSTpull-down and Western-Blot method to detect the activity level of Rap1
HEK293 cell is inoculated in 100cm
2double dish, when cell grows to 80% density, utilizes liposome transfection plasmid Rap1V12 (rap1 genome constitutive active form, expression product is equivalent to Rap1-GTP).Collecting cell after 24h, first washes once with PBS, then uses IPbuffer lysate cell lysis on ice, and the centrifugal 10min of 12000rpm, gets supernatant, leaves and takes wherein 20% in contrast.The cell conditioned medium of transfection Rap1V12 is divided into two parts, the glutathione sepharose beads of GST empty carrier and GST-RalGDS-RBD mixes with coupling respectively, put 4 DEG C of DLs on shaking table and turn 1h, 4 DEG C of 6000rpm are centrifugal, wash three times with IP lysate, finally blot all remaining liqs in Ep pipe with microsyringe, add 30 μ lSDS-PAGEloadingbuffer, boil 10min, detect target Rap1 albumen finally by western-blot.As shown in Figure 5, wherein using GST empty carrier as negative control, GST-RalGDS-RBD's result can interact with the Rap1-GTP of HEK293 cells, and this interaction has very strong specificity.
Utilize liposome transfection plasmid Rap1V12 (rap1 genome constitutive active form, expression product is equivalent to Rap1-GTP) or Rap1N17 (the shaping inactive forms of rap1 genome, expression product is equivalent to Rap1-GDP) in HEK293 cell.Collecting cell after 24h, first washes once with PBS, then uses IP lysate cell lysis on ice, and the centrifugal 10min of 12000rpm, gets supernatant, leaves and takes wherein 20% in contrast.The glutathione sepharose beads of coupling GST-RalGDS-RBD mixes with the cell conditioned medium of transfection Rap1V12 and the cell conditioned medium of transfection Rap1N17 respectively, put 4 DEG C of DLs on shaking table and turn 1h, 4 DEG C of 6000rpm are centrifugal, three times are washed with IP lysate, finally blot all remaining liqs in Ep pipe with microsyringe, add 30 μ lSDS-PAGEloadingbuffer, boil 10min, detect target Rap1 albumen finally by western-blot.As shown in Figure 6, GST-RalGDS-RBD's result can interact with the Rap1-GTP of HEK293 cells, but the Rap1-GDP of discord HEK293 cells combines.Ultimate principle of the present invention
Small molecular G protein Rap1 activity detection kit is based on glutathione s-transferase (glutathioneS-transferase, GST) affinity chromatography and GSTpull-down method.First recombinant technique is utilized glutathione s-transferase and the RalGDS-RBD containing Rap1 calmodulin binding domain CaM (Rap1bindingdomain, RBD) to be merged.The affine combination of glutathione (Glutathione, GTH) on carrier by GST and immobilization of GST-RalGDS-RBD fusion, also can by Rap1 calmodulin binding domain CaM in conjunction with Rap1-GTP.When GST-RalGDS-RBD fusion is affine be combined in glutathione solidification Ago-Gel pearl time, this solid phase carrier can get off Rap1-GTP pull-down from cell or tissue lysate.After IP lysate washes away the albumen be not combined with solid phase carrier, then make the protein sample be separated for sds polyacrylamide gel electrophoresis.Utilize the detection method of WesternBlot, use Rap1 antibody, get final product the activity level of Rap1 in specific detection pathological tissue or cell.
The advantage of the inventive method:
1. the detection method of the present invention's employing is simple to operate, is easy to standardization.
2. the GST-RalGDS-RBD fusion purity of the present invention's employing is high, and specificity is good.
3. the inhibitors of phosphatases potpourri that the present invention adopts can suppress Rap1-GTP to degrade well, thus ensures the authenticity of experimental result.
The composition of kit of the present invention:
1.GST-RalGDS-RBD fusion
Specification: 600 μ g, can for 30 pull-down experiments, protein purification concentration >75% (shown in Fig. 4).
Preservation condition: be dissolved in 50mMTris-HCl, 10% glycerine, in 0.02%NaN3, is kept at-80 DEG C.Purposes: GST-RalGDS-RBD fusion, also can by Rap1 calmodulin binding domain CaM in conjunction with Rap1-GTP by GST and the affine combination of the glutathione of immobilization on carrier.
1.IPbuffer
Specification: 200ml
Purposes: IPbuffer is used for pathological tissue or lysis, is kept at 4 DEG C.
Note: add protease inhibitors cocktail (1:500) and inhibitors of phosphatases during use.
1.Washingbuffer
Washingbuffer, for washing glutathione sepharose beads, is kept at 4 DEG C.
Note: add protease inhibitors cocktail (1:500) and inhibitors of phosphatases during use.
2. glutathione sepharose beads
Specification: 450 μ l, are kept at 4 DEG C.
Purposes: as solid phase carrier, in conjunction with GST-RalGDS-RBD fusion.
3.SDS-PAGEloadingbuffer
Specification: 1ml, room temperature preservation.
Purposes: containing 4%SDS (w/v), 2%glycerol and 0.05% bromophenol blue, for albumen sample preparation, be kept at 4 DEG C.
4.Rap1 antibody
Specification: anti-Rap1 (50 μ l, Sc-65, SantaCruz company)
Purposes: detect for Westernblot.
5. proteinase inhibitor C ocktail (50 μ l), is kept at-20 DEG C.
6. inhibitors of phosphatases 500mMNaF (100 μ l), 100mMNa3VO4 (100 μ l), be kept at 4 DEG C.Note: NaF working concentration 10mM, Na3VO4 working concentration 1mM, plays and suppresses the dephosphorylized effect of Rap1-GTP.
The using method of this kit
1. in 1.5mlEp pipe, add 15 μ l glutathione sepharose beads;
2. add the washingbuffer of 0.5ml precooling, 4 DEG C of 6000rpm are centrifugal, wash three times;
3. abandon supernatant, the resuspended glutathione sepharose beads of IPbuffer of 200 μ l precoolings;
4. add 20 μ lGST-RalGDS-RBD fusions, 4 DEG C of shaking tables hatch 60min, note: solution needs shake;
5. add the washingbuffer of 0.5ml precooling, 4 DEG C of 6000rpm are centrifugal, wash three times;
6. abandon supernatant, add tissue or cell lysate, 4 DEG C of shaking tables hatch 60min; Note: pathological tissue IPbuffer cracking, need fully to grind 5min, then 13000rpm is centrifugal, gets supernatant simultaneously.Cell, with after IPbuffer cracking, also needs 13000rpm centrifugal, then gets supernatant.
7. add the washingbuffer of 0.5ml precooling, 4 DEG C of 6000rpm are centrifugal, wash three times;
8. abandon supernatant, add 10 μ lSDS-PAGEloadingbuffer, boiling water boiling protein sample 5-10min;
9.Westernblot detects, anti-Rap1 antibody, recommends primary antibodie concentration (1:2000), two anti-concentration (1:20000).
Accompanying drawing explanation
Fig. 1 pGEX-6P-1 plasmid map and restriction endonuclease sites.
Fig. 2 agarose gel electrophoresis detects the about 410bp of BamHI and EcoRI endonuclease bamhi (left figure) and the about 4.9kb of carrier segments (right figure).
(non-RED sector is identical sequence, and 1-387 is theoretical sequence for Fig. 3 DNAssist software comparison object sequencing fragment result and theoretical sequence; 1-500 is sequencing sequence).
The SDS-PAGE coomassie brilliant blue staining figure of Fig. 4 GST-RalGDS-RBD fusion.
The combination of Fig. 5 GST-RalGDS-RBD fusion and Rap1-GTP, GST is not in conjunction with Rap1-GTP.
Fig. 6 GST-RalGDS-RBD fusion in conjunction with Rap1-GTP, and not in conjunction with Rap1-GDP.
Fig. 7 detects the active process flow diagram of Rap1.
After Fig. 8 NGF stimulates PC12 cell, Rap1 activity level in this kit detection cell.
After Fig. 9 NGF stimulates PC12 cell, this kit detects Rap1 activity level in NG108-15 and HEK293 cell.
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment one: utilize this kit to detect Rap1 activity level in PC-12 cell
Concrete implementation step is as follows:
1. the cell conditioned medium liquid of preparation containing Rap1-GTP
Bibliographical information nerve growth factor (NGF, 50ng/mL) is had to stimulate PC-12 cell that Rap1 can be caused to activate.In NGF stimulation test, inoculating cell is in 35cm
2in double dish, serum starvation 16h when cell grows to 80% density.Processed group cell final concentration is that the NGF of 50ng/mL stimulates 10min, and does negative contrast.Stimulate after terminating, once, the IPbuffer utilizing this kit to provide is on ice after cell lysis, and the centrifugal 10min of 12000rpm, gets supernatant for subsequent use for PBS washed cell, leaves and takes wherein 20% in contrast.
2. according to using method 1-5 described in kit of the present invention, process glutathione sepharose beads.
3. according to using method 6 described in kit of the present invention, in the glutathione sepharose beads processed, add the supernatant described in this implementation step 1,4 DEG C of shaking tables hatch 60min.
4. according to using method 7-8 of the present invention, processing sample.
5.Westernblot method detects Rap1 activity level in cell (shown in Fig. 8).
Embodiment two: utilize this kit to detect Rap1 activity level in NG108-15 and HEK293 cell
Concrete implementation step is as follows:
1. the cell conditioned medium liquid of preparation containing Rap1-GTP
Bibliographical information NG108-15 cell background is had to there is the Rap1 albumen activated, the Rap1 then do not activated in HEK293 cell.Inoculating two kinds cell is in 100cm respectively
2in double dish, when cell grows to 90% density, PBS washed cell twice, the IPbuffer utilizing this kit to provide is on ice after cell lysis, and the centrifugal 10min of 12000rpm, gets supernatant for subsequent use, leaves and takes wherein 20% in contrast.
2. according to using method 1-5 described in kit of the present invention, process glutathione sepharose beads.
3. according to using method 6 described in kit of the present invention, in the glutathione sepharose beads processed, add the supernatant described in this implementation step 1,4 DEG C of shaking tables hatch 60min.
4. according to using method 7-8 of the present invention, processing sample.
Westernblot method detects Rap1 activity level in NG108 and HEK293 cell (shown in Fig. 9).
Claims (6)
1. detect a method for small molecular G protein Rap1 activity, its concrete steps are as follows:
Step 1 builds GST-RalGDS-RBD prokaryotic expression plasmid
In inquiry ncbi database, hRalGDS gene pairs answers the coded sequence of 798-885 amino acids residue, the multiple clone site (shown in Fig. 1) of contrast prokaryotic expression carrier pGEX-6P-1, BamHI and EcoRI site is wherein selected to design upstream primer (5 '-CGCGGATCCGCGctgccgctctacaacc-3 ') and downstream primer (5 '-CCGGAATTCCGGtcagaagatgcccttggcaa-3 ') as restriction enzyme site, with people liver library for masterplate, pcr amplification obtains object fragment; After agarose gel electrophoresis is separated, reclaim kit with gel reclaim, the object fragment reclaimed and pGEX-6P-1 empty carrier are respectively after BamHI and EcoRI double digestion, again through agarose gel electrophoresis separation and recovery, the object fragment of recovery and carrier mix with the ratio of 5:1; Then T is used
4dNA ligase spends the night in 16 DEG C of connections, and transform DH5 α Escherichia coli by this system subsequently, be coated with flat board and the positive colony selected wherein, selected positive colony obtains the object fragment of about 418bp after restriction enzyme is cut; The coded sequence that GST-RalGDS-RBD prokaryotic expression plasmid checks order in the object fragment and ncbi database that obtain is completely the same;
The prokaryotic expression of step 2GST-RalGDS-RBD fusion and purifying
With the GST-RalGDS-RBD Plastid transformation BL21 Escherichia coli of above-mentioned structure, transfer in 100mLLB nutrient culture media by 1%, 37 DEG C of 180r/min shaken cultivation are to exponential phase (D
600nm=0.6); IPTG is added subsequently, 20 DEG C of 180r/min vibration overnight induction by 100 μMs/L final concentration; Next day 4 DEG C of 4000rpm10min collected by centrifugation bacterium liquid, by resuspended for the Escherichia coli 30mL damping fluid PB [containing 0.5mM/LDTT, 2 μ g/mLAprotimin, 2 μ g/mLLeupeptin, 1mM/LPMSF, 1mM/LNa3VO4,10mM/LNaF in 30mLPBS] obtained, then high pressure is broken, and ultrasound wave is broken further subsequently; 4 DEG C of centrifugal pyrolysis products of 13000rpm10min, get supernatant, the supernatant that the 300 μ L glutathione sepharose beads in advance through overbalance obtain after centrifugal is mixed, 4 DEG C of DLs turn 2h, then the PB buffer solution glutathione sepharose beads of precooling, utilize elution buffer [5ml fills a prescription: 250 μ L1M/LTris-Cl, ph7.6,4.75mL ultrapure water, 0.0154g reduced glutathione] that GST-RalGDS-RBD is washed from gel beads; SDS-PAGE coomassie brilliant blue staining method is finally utilized to detect the GST-RalGDS-RBD fusion of purifying;
Step 3 uses GSTpull-down and Western-Blot method to detect the activity level of Rap1
HEK293 cell is inoculated in 100cm
2double dish, when cell grows to 80% density, utilizes liposome transfection plasmid Rap1V12 (rap1 genome constitutive active form, expression product is equivalent to Rap1-GTP); Collecting cell after 24h, first washes once with PBS, then uses IPbuffer lysate cell lysis on ice, and the centrifugal 10min of 12000rpm, gets supernatant, leaves and takes wherein 20% in contrast; The cell conditioned medium of transfection Rap1V12 is divided into two parts, the glutathione sepharose beads of GST empty carrier and GST-RalGDS-RBD mixes with coupling respectively, put 4 DEG C of DLs on shaking table and turn 1h, 4 DEG C of 6000rpm are centrifugal, wash three times with IP lysate, finally blot all remaining liqs in Ep pipe with microsyringe, add 30 μ lSDS-PAGEloadingbuffer, boil 10min, detect target Rap1 albumen finally by western-blot; As shown in Figure 5, wherein using GST empty carrier as negative control, GST-RalGDS-RBD's result can interact with the Rap1-GTP of HEK293 cells, and this interaction has very strong specificity.
2. detect a kit for small molecular G protein Rap1 activity, comprising:
GST-RalGDS-RBD fusion
Specification: 600 μ g, can for 30 pull-down experiments, protein purification concentration >75% (shown in Fig. 4);
Preservation condition: be dissolved in 50mMTris-HCl, 10% glycerine, in 0.02%NaN3, is kept at-80 DEG C;
IPbuffer
Specification: 200ml
Purposes: IPbuffer is used for pathological tissue or lysis, is kept at 4 DEG C;
Washingbuffer
Washingbuffer, for washing glutathione sepharose beads, is kept at 4 DEG C;
Glutathione sepharose beads
Specification: 450 μ l, are kept at 4 DEG C;
SDS-PAGEloadingbuffer
Specification: 1ml, room temperature preservation;
Rap1 antibody
Specification: anti-Rap1 (50 μ l, Sc-65, SantaCruz company)
Proteinase inhibitor C ocktail (50 μ l), is kept at-20 DEG C;
Inhibitors of phosphatases 500mMNaF (100 μ l), 100mMNa3VO4 (100 μ l), be kept at 4 DEG C.
3. a plasmid, it is under suitable isopropylthiogalactoside (IPTG) inductive condition, can at expression in escherichia coli GST-RalGDS-RBD fusion.
4. plasmid as claimed in claim 3, is characterized in that the coded sequence of answering 798-885 amino acids residue (Rap1 binding structural domain) containing hRalGDS gene pairs.
5. a fusion GST-RalGDS-RBD, is characterized in that being made up of glutathione s-transferase and the RalGDS-RBD containing Rap1 calmodulin binding domain CaM.
6. albumen as claimed in claim 5, is characterized in that, by GST and the affine combination of the glutathione of immobilization on carrier, and also can by Rap1 calmodulin binding domain CaM in conjunction with Rap1-GTP.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2004005550A2 (en) * | 2002-07-04 | 2004-01-15 | Fujisawa Pharmaceutical Co., Ltd. | Method of screening for antidiabetic agents |
| WO2005003783A1 (en) * | 2003-06-26 | 2005-01-13 | Babraham Institute | Methods for identifying compounds interacting with small membrane-bound gtp-ases |
| CN102692505A (en) * | 2011-03-21 | 2012-09-26 | 中国医学科学院肿瘤研究所 | Improved co-immunoprecipitation technical method |
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2015
- 2015-11-13 CN CN201510776243.9A patent/CN105445472A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004005550A2 (en) * | 2002-07-04 | 2004-01-15 | Fujisawa Pharmaceutical Co., Ltd. | Method of screening for antidiabetic agents |
| WO2005003783A1 (en) * | 2003-06-26 | 2005-01-13 | Babraham Institute | Methods for identifying compounds interacting with small membrane-bound gtp-ases |
| CN102692505A (en) * | 2011-03-21 | 2012-09-26 | 中国医学科学院肿瘤研究所 | Improved co-immunoprecipitation technical method |
Non-Patent Citations (2)
| Title |
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| ARIEL F.CASTRO ET AL.: "Measuring Ras-family GTP levels in vivo—running hot and cold", 《METHODS》 * |
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Application publication date: 20160330 |