CN101775103B - Preparation method of protein molecule engram film - Google Patents
Preparation method of protein molecule engram film Download PDFInfo
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- CN101775103B CN101775103B CN200910273432A CN200910273432A CN101775103B CN 101775103 B CN101775103 B CN 101775103B CN 200910273432 A CN200910273432 A CN 200910273432A CN 200910273432 A CN200910273432 A CN 200910273432A CN 101775103 B CN101775103 B CN 101775103B
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Abstract
The invention discloses a preparation method of a protein molecule engram film, comprising the following steps: 1. pretreatment of carrier sheet glass used for preparing a protein molecule engram film: putting a cover glass into a beaker, adding cleaning agent and deionized water, and processing by ultrasound; 2. preparation of the solution of template protein, functional monomer and crosslinking monomer: preparing template protein solution by phosphate buffer, adding water-solubility functional monomer, crosslinking monomer and initiator, and evenly mixing; 3. carrying out polymerization between sheet glasses to form a polymer film; 4. elution of template protein molecule: putting the molecule engram film into a beaker, adsorbing eluent in the beaker, removing the template protein molecule, and obtaining an engram hole matched with the template protein molecule. In the engram process of the invention, protein has small possibility of denaturation, and polymerization condition is simple and moderate. The prepared molecule engram film can be developed into a developing chip capable of detecting protein.
Description
Technical field
The present invention relates to the molecular imprinting field, more specifically relate to a kind of preparation method of protein molecule engram film.Be suitable for the macromolecular molecular imprinting of all water-soluble biologicals.Utilize the molecular engram film of this method preparation that target protein is had certain special identification function, can develop into and substitute the proteinic analysed preparation of detection of antibodies.
Background technology
Molecular imprinting is a kind of synthetic artificial antibody's a technology, and its cardinal principle is: at first select to form with template molecule the function monomer of covalent linkage or non covalent bond, form mixture with template molecule, form cross-linked polymer with the linking agent copolymerization then.Behind the template molecule wash-out, in polymkeric substance, stay the hole that shape, size and surface functional group with template molecule are complementary, these cavity energy selectivity recognition template molecules.Adopt molecular imprinting, preparation is processed the molecular recognition elements of alternative antibody to the molecular engram film that the target protein molecule has excellent recognition capability, is a new research direction of biochip and field of biosensors in recent years.
At present, the preparation method of the normal protein molecule engram film that adopts has following several kinds: one, directly on sensor surface (electrode, QCM substrate and SPR substrate), carry out mass polymerization and form the western blotting film.Like the honest and poor Biosens.Bioelectron.2006 that waits of Yao of Hunan University, 21:1244-1251 is through the method for sol-gel and electroless plating, the molecular engram film of the human serum albumin discerned for preparing in piezoelectric quartz crystal-gold electrode surfaces.The signal of its transmitter can increase along with the increase of tested protein concentration, and weak point is that Selectivity of Sensor is not ideal enough.Two, the identical small peptide of employing and proteantigen decision group composition, as template molecule, but the molecular engram film of synthetic identification of protein in the plane.The artificial antibody's film Anal.Chem. that detects dengue fever virus, 2005,77:5140-5143 have been prepared like employing polypeptide blottings such as Tai.But in the work of Tai, measure circulation ratio relatively poor (CV is at 10%-32%); Reason is that the template molecule that adopts is the small peptide that has only several amino acid; Do not possess Three Dimensions Structure; And the three-dimensional structure of polypeptide is the main foundation of Ag-Ab identification, so the blotting membrane of this method preparation is difficult to hold to the recognition efficiency of protein molecule.Three, stamped method is fixed on formation " protein seal " on a certain substrate with protein template, closely contacts with the polymer monomer layer, and polyreaction is carried out in very thin liquid layer.This method can make ultra-thin molecular engram film.Like [10-12] such as Chou protein template is adsorbed on the sheet glass, the sheet glass that has been coated with function monomer and cross-linking monomer with another piece closely contacts, and causes through UV-light; Polyreaction is carried out in the interlayer of two blocks of sheet glass, make protein molecular blotting membrane Biosens.Bioelectron., 2006; 22:355-363; Biosens.Bioelectron., 2006,22:534-543.Shi etc. with protein adsorption in hydrophilic, atomically flat mica surface; Then a disaccharides thin layers of molecules is covered on the protein of absorption; Through a large amount of hydrogen bonds and protein complexing, again with a smooth fluoro-containing copolymer film through the light emitting discharge plasma body and glycan molecule is crosslinked deposits, obtain having molecular engram film Nature with the nanometer hole of protein form fit; 1999,398:593-597.Yet; Shi and Chou etc. are with method direct fixing protein molecules on solid surface of physical adsorption; Not only can cause protein aggregation, and cause the variation on its configuration easily, cause protein denaturation; The trace hole of coming out can be poor to the recognition capability of natural structure molecule like this, also caused the ununiformity of molecular engram film.Four, self-assembly method, Britt etc. self-assemble to protein template molecule and three kinds of phosphatide on the solid surface jointly.The unimolecular layer that phospholipid molecule forms on solid surface; Avoided the direct absorption of protein at solid surface; The phosphatide that wherein has positive charge is adsorbed on electronegative protein molecule on the phospholipid layer through electrostatic interaction; And the phospholipid molecule that is connected with the PEG chain has stoped the gathering Biotechnol.Prog.2006 between protein molecule, 22:150-155.Though aforesaid method helps the formation in unit molecule trace hole, also avoided the distortion of template molecule to a certain extent.But owing to do not adopt cross-linking monomer, the stability in trace hole there is certain influence.
To sum up, though forefathers are accumulating many valuable experience aspect the preparation of protein molecule engram film, yet; The protein volume is big; Complex structure (its surface shape is irregular, and functional group is many), the easy deformation sex change; Unstable in organic solvent, work has brought very big difficulty to western blotting.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of protein molecule engram film; This method is easy, economical; This method adopts protein electrophorese glue raw material propylene acid amides commonly used as function monomer; The mixing solutions that " sandwich " method of employing is about to template albumen, function monomer, cross-linking monomer and initiator drips between two blocks of sheet glass, and polyreaction is carried out between two blocks of sheet glass, forms the protein molecule engram film.Acrylic amide is the water-solubility function monomer, has good consistency with protein, is difficult for causing protein denaturation in the polymerization process, in addition, and the crosslinking polymerization mild condition of acrylic amide, the also molecular imprinting of suitable biomacromolecule.
The present invention realizes through following technical scheme:
A kind of preparation method of protein molecule engram film the steps include:
(1), the pre-treatment of the carrier sheet glass of preparation protein molecule engram film:
Deckglass is put into beaker, the clean-out system (washing powder of various brands and the cleaning agent that can be used for glassware washing) that adds a certain amount of 1～5 gram in 300～500mL deionized water, ultrasonic 5～20min; Another beaker is put in the deckglass taking-up, after 3～5 flushings of deionized water, in the 1mol/L sodium hydroxide solution, soaked and ultrasonic 5～20min; Take out deckglass, to remove the sodium hydroxide solution that remains on the deckglass, after 3～5 flushings, add the ultrasonic 5～20min of deionized water with deionized water rinsing; Ionized water is poured out, added 1mol/L HCl solution soaking, ultrasonic 5～20min; Take out deckglass, to remove the HCl solution that remains on the slide, after 3～5 flushings, add the ultrasonic 5～20min of deionized water with deionized water rinsing; Above-mentioned deckglass is taken out, dry, subsequent use.
(2), the preparation of template albumen, function monomer and cross-linking monomer solution:
With phosphate buffered saline buffer preparation finite concentration 10
-6～10
-8The BHb solution of M, take by weighing a certain amount of (require with the proteic mole ratio of template reach more than 400: 1) water-solubility function monomer as: acrylic amide, USAF RH-1, vinylformic acid, methylacrylic acid etc. are added in the 1mL protein solution mixing; 4 ℃ of placements are spent the night in refrigerator; Add N again, N '-methylene-bisacrylamide, ultrasonic degas 5～10min; Behind logical nitrogen 5～10min; Add initiator 5～10 μ L successively, (mol ratio that makes itself and function monomer is between 500～1000) and catalyzer (tetramethyl-diethylamine) 1 μ L, mixing;
(3), between two blocks of sheet glass, carry out polyreaction:
Set by step (2) with protein template molecule, function monomer, cross-linking monomer and initiator mixing after, draw their mixed liquid of 30～50mL with pipettor, slowly drip on slide glass; Covered; Drop is covered entirely, guarantees no bubble, polymerization 2-3 hour as far as possible; Open deckglass after the film forming gently, obtain the acrylamide polymer film the same with the deckglass area.
(4), wash-out template protein molecular:
(MIPM) puts into small beaker with molecular engram film, draws the 2mL elutriant at every turn and adds wherein, shakes and surveys its ultraviolet-visible light absorption value after 3-4 hour, till detecting less than absorption peak, rinses out the eluent that remains on the film with deionized water at last.Through surveying proteic content in the elutriant, judge the elution efficiency of elutriant.
(5), prepare non-blotting membrane (NIPM) by (1)-(4) step, promptly in the process of preparation polymeric film, do not add the template protein molecular.
(6), molecular engram film is to the method for calculation of the adsorptive capacity of target protein:
MIPM and the NIPM that will pass through elution process put into respectively and are added with 6 orifice plates that heavily combine the template protein solution; (15～60min) utilize ultraviolet/visible spectrophotometer to measure the absorbance that heavily combines the template protein solution in 6 orifice plates, calculate molecular engram film (MIPM) and non-molecular engram film (NIPM) to proteic adsorptive capacity according to the adsorptive capacity formula at regular intervals.Adsorptive capacity: Q
Inhale=(CO-C
Surplus) * V/S, wherein Q
InhaleBe MIPM and the proteic adsorptive capacity of NIPM absorption template; C
OAttach most importance to and combine the concentration of template protein solution; C is surplus for combining 2 hours or heavily combined in 6 orifice plates after the longer time concentration of template protein solution; V attaches most importance to and combines the volume of template protein solution; S is a membrane area.
(7), the selective adsorption evaluation method of molecular engram film:
MIPM and NIPM put into respectively fill the different sorts protein solution respectively: HEL; Bovine serum albumin, myohaemoglobin is in 6 orifice plates of BHb; Leave standstill and place after 4 hours; Utilize ultraviolet/visible spectrophotometer to measure the concentration of the heavy conjugated protein solution of different sorts in 6 orifice plates, calculate adsorptive capacity, estimate blotting membrane to the proteic recognition performance of template through the value of the trace factor (imprinting factor) α.α value (imprinting factor)=Q
MIPM inhales/ Q
NIPM inhales, in the formula: Q
MIPM inhalesBe the loading capacity of blotting membrane to certain protein soln; Q
NIPM inhalesBe the loading capacity of non-blotting membrane to certain protein soln.
The carrier of described blotting membrane can be tolerance such as simple glass, a silica glass or mica washings, not with the material of polymeric solution reaction.
Described template albumen can be water-soluble and with function monomer, all compatible albumen of cross-linking monomer, like in BHb, the myohaemoglobin any one.
Described all functions monomer and cross-linking monomer are water miscible, and can not make template albumen generation sex change.Comprise water-solubility function monomer acrylic amide or USAF RH-1 or acrylic or methacrylic acid a kind of or two to four kinds and cross-linking monomer N wherein, N '-methylene-bisacrylamide.
Described elutriant can be with buffered soln, salts solution, alkaline solution and the denaturing agent etc. of template albumen wash-out from the blotting membrane for all.Wherein a kind of of phosphate buffer soln or NaCl salts solution or NaOH alkaline solution or the denaturing agent sodium lauryl sulphate that comprises various concentration or two to four kinds.
Described initiator is can cause polyreaction but do not make all initiators of template albumen generation sex change comprise thermal initiator and light trigger.Comprise wherein a kind of such as Potassium Persulphate, ammonium persulphate, Thiocarb.
Described catalyzer is: the tetramethyl-diethylamine.
Adopt the inventive method can prepare the molecular engram film that target protein is had selective adsorption.The result shows that acrylic amide is a kind of function monomer for preparing the western blotting film preferably, and in film-forming process, sex change does not take place template albumen, and therefore, the hole that forms on the molecular engram film has recognition capability to target protein.The inventive method is simple than document reported method; Function monomer is easy to get; Reaction conditions is gentle, is a kind of easy, economic method for preparing the biomacromolecule molecular engram film, and the protein molecule engram film of this method preparation can be developed into and detects proteinic chip.Experimental data shows that blotting membrane (MIPM) is 0.21～0.22nmol/cm to the adsorptive capacity of the adsorptive capacity of BHb
2Apparently higher than the adsorptive capacity 0.09～0.1nmol/cm of non-blotting membrane to oxyphorase
2Blotting membrane is followed successively by 0.08nmol/cm to the adsorptive capacity of BSA, HEL, myohaemoglobin and the BHb of same concentrations
2, 0.1nmol/cm
2, 0.14nmol/cm
2And 0.21nmol/cm
2, show that blotting membrane is the highest to the adsorptive capacity of template albumen-BHb.
Description of drawings
The preparing method's of a kind of protein molecule engram film of Fig. 1 blotting membrane concerns synoptic diagram to proteic adsorptive capacity of template and adsorption time.
The preparing method's of a kind of protein molecule engram film of Fig. 2 different proteic absorption situation synoptic diagram.
Embodiment
Embodiment 1:
A kind of preparation method of protein molecule engram film the steps include:
One, the processing of carrier-sheet glass matrix:
Experiment adopts " sandwich " sandwich assay to prepare molecular engram film, and (24 * 24mm and 18 * 18mm) is as " carrier " to adopt two kinds of slide glasss that vary in size.The pre-treatment of carrier slide, concrete steps are following:
(1) deckglass is put into beaker, add clean-out system (any model washing powder or liquid cleaning agent) and 300～500mL deionized water of 1～5 gram, ultrasonic 5～20min;
(2) another beaker is put in the deckglass taking-up, after 3～5 flushings of deionized water, in the 1mol/L sodium hydroxide solution, soaked and ultrasonic 5～20min;
(3) take out deckglass, to remove the sodium hydroxide solution that remains on the deckglass, after 3～5 flushings, add the ultrasonic 5～20min of deionized water with deionized water rinsing.
(4) ionized water is poured out, added the 1mol/LHCl solution soaking, ultrasonic 5～20min.
(5) take out deckglass, to remove the HCl solution that remains on the slide, after 3～5 flushings, add the ultrasonic 5～20min of deionized water with deionized water rinsing.
(6) above-mentioned deckglass is taken out, dry, subsequent use.
Two, the preparation of blotting membrane:
With the hemoglobin solutions of PBS (0.01mol/L pH6.4) preparation 0.168 μ mol/L, add acrylic amide, the mol ratio that makes itself and oxyphorase shakes up greater than 400, and 4 ℃ of placements are spent the night in refrigerator.Add N again, N '-methylene-bisacrylamide, making the mol ratio of itself and function monomer is 1: 40, ultrasonic degas 5min, logical again nitrogen 5min.Toward 5mL oxyphorase, acrylic amide and N, add ammonium persulphate and the 1 μ L tetramethyl-diethylamine of 10 μ L 0.1M in the mixing solutions of N '-methylene-bisacrylamide successively, behind the mixing; Draw 50ul with pipettor; Slowly drip on slide glass, covered is covered drop entirely; Guarantee no bubble, polymerization 2-3 hour as far as possible.Open deckglass after the film forming gently, obtain the polymeric film the same with the deckglass area.The preparation process of non-blotting membrane and blotting membrane basic identical just do not add the template molecule BHb in the polymerization process.
Three, the wash-out of template molecule:
(MIPM) puts into small beaker with molecular engram film; The each 2mL of absorption elutriant adds wherein;, shaking table surveys its ultraviolet-visible light absorption value after shaking 3-4 hour with the ultraviolet-visible spectrophotometer; Till detecting, rinse out the elutriant that remains on the film with deionized water at last less than absorption peak.Described elutriant comprises: 1M NaCl, 0.1M PBS, 1%SDS-10%HAc.Experimental result shows that the elution amount with 1%SDS-10%HAc solution is 2.9nmol to the maximum, and elution efficiency is 47%, and other elutriant elute effects of comparing are best.0.1mol/LPBS solution takes second place, elution amount is 1.4nmol, and elution efficiency is 17%; And 0.1M NaCl solution is minimum to proteic elution amount, and elution amount is for being 0.9nmol, and elution efficiency is 11%.Non-blotting membrane is carried out same elution process.
Four, molecular engram film is to the absorption of target protein:
MIPM and the NIPM that will pass through elution process put into respectively and are added with 6 orifice plates that 1.125 μ mol/L heavily combine the template protein solution; Every utilize ultraviolet/visible spectrophotometer to measure heavily to combine in 6 orifice plates template protein solution absorbance, calculate MIPM and NIPM to proteic adsorptive capacity according to the adsorptive capacity formula at a distance from 30min.Adsorptive capacity: Q
Inhale=(C
O-C
Surplus) * V/S, wherein Q
InhaleBe MIPM and the proteic adsorptive capacity of NIPM absorption template; C
OAttach most importance to and combine the concentration of template protein solution; C
SurplusFor heavily combining the concentration of template protein solution in 6 orifice plates after combination for some time; V attaches most importance to and combines the volume of template protein solution; S is a membrane area.Fig. 1 shows: in 2.5h, blotting membrane (MIPM) obviously increases along with the increase of time the adsorptive capacity of BHb, is 0.21～0.22nmol/cm to the proteic adsorptive capacity of template when blotting membrane reaches the Static Adsorption balance
2
Non-blotting membrane (NIPM) also has certain adsorptive capacity to BHb, and along with the increase of time also has increase to a certain degree, but adsorptive capacity and increasing degree are starkly lower than blotting membrane, and NIPM is 0.09～0.1nmol/cm to the adsorptive capacity of BHb
2
Five, the selective adsorption evaluation of molecular engram film:
MIPM and NIPM put into fill the different sorts protein solution respectively: HEL, bovine serum albumin, myohaemoglobin, in 6 orifice plates of BHb, protein concentration is 1.125 μ mol/L.Leave standstill and place after 4 hours; Utilize ultraviolet/visible spectrophotometer to measure the concentration of the heavy conjugated protein solution of different sorts in 6 orifice plates; Calculate adsorptive capacity, estimate blotting membrane to the proteic recognition performance of template through the value of the trace factor (imprinting factor) α.α value (imprinting factor)=Q
MIPM inhales/ Q
NIPM inhales, in the formula: Q
MIPM InhaleBe the loading capacity of blotting membrane to certain protein soln; Q
NIPMInhale and be the loading capacity of non-blotting membrane certain protein soln.Fig. 2 shows that blotting membrane is followed successively by 0.08nmol/cm to the adsorptive capacity of BSA, HEL, myohaemoglobin and the BHb of same concentrations
2, 0.1nmol/cm
2, 0.14nmol/cm
2And 0.21nmol/cm
2Blotting membrane is the highest to the adsorptive capacity of template albumen-BHb, and blotting membrane is respectively 1.2,1.3,1.7,2.5 to the α value of BSA, N,O-Diacetylmuramidase, myohaemoglobin and BHb, explains that MIPM has certain selectivity to BHb.Non-blotting membrane is to BSA, N,O-Diacetylmuramidase, myohaemoglobin and little to the adsorptive capacity difference of template protein B HB solution, at 0.07-0.08nmol/cm
2Between, show that non-blotting membrane does not have selectivity to albumen.The result shows that blotting membrane has the selection identity that more has than non-blotting membrane.
Embodiment 2:
A kind of preparation method of protein molecule engram film the steps include:
One, the processing of carrier-sheet glass matrix:
The processing of carrier-slide matrix is identical with embodiment 1.
Two, the preparation of blotting membrane:
Hemoglobin solutions with PBS (0.01mol/L pH6.4) preparation 0.084 μ mol/L.Add function monomer acrylic amide and vinylformic acid (mol ratio 1: 1), the mole ratio of total mole number and oxyphorase that makes function monomer shakes up greater than 400, and 4 ℃ of placements are spent the night in refrigerator.Add N again, N '-methylene-bisacrylamide, making the mol ratio of itself and function monomer is 1: 40, ultrasonic degas 5min, logical again nitrogen 5min.Add ammonium persulphate and the 1 μ L tetramethyl-diethylamine of 10 μ L0.1M, behind the mixing, draw 50 μ L with pipettor, slowly drip on slide glass, covered is covered drop entirely, guarantees no bubble, polymerization 2-3 hour.Open deckglass after the film forming gently, obtain the polymeric film the same with the deckglass area.The preparation process of non-blotting membrane and blotting membrane basic identical just do not add the template molecule BHb in the polymerization process.
Three, the wash-out of template molecule:
The step of the wash-out of template molecule is identical with embodiment 1.The elutriant that adopts is 1%SDS-10%HAc.
Four, molecular engram film is to the absorption of target protein:
Molecular engram film is identical with embodiment 1 to the adsorption step of target protein, and experimental result shows that molecular engram film is 0.380nmol/cm to the adsorptive capacity of oxyphorase
2, be higher than the adsorptive capacity of non-blotting membrane to oxyphorase.
Embodiment 3:
One, the processing of carrier-sheet glass matrix:
The processing of carrier-slide matrix is identical with embodiment 1.
Two, the preparation of blotting membrane:
With the hemoglobin solutions of PBS (0.01mol/L pH6.4) preparation 0.042 μ mol/L, add methylacrylic acid and acrylic amide (mol ratio 1: 1), the mol ratio that makes itself and oxyphorase shakes up greater than 400, and 4 ℃ of placements are spent the night in refrigerator.Add N again, N '-methylene-bisacrylamide, N, the mol ratio of N '-methylene-bisacrylamide and function monomer is 1: 40.Ultrasonic degas 5min, logical again nitrogen 5min.Add 10 μ L0.1M Sodium Persulfates and tetramethyl-diethylamine 1 μ L, behind the mixing, draw 50ul with pipettor and slowly drip on slide glass, covered is covered drop entirely, guarantees no bubble, polymerization 2-3 hour as far as possible.Open deckglass after the film forming gently, obtain the polymeric film the same with the deckglass area.The preparation process of non-blotting membrane and blotting membrane basic identical just do not add the template molecule BHb in the polymerization process.
Three, the wash-out of template molecule:
The elution step of template molecule is identical with embodiment 2.
Four, molecular engram film is to the absorption of target protein:
Molecular engram film is identical with embodiment 1 to the adsorption step of target protein, and experimental result shows that molecular engram film is 0.130nmol/cm to the adsorptive capacity of oxyphorase
2, be higher than the adsorptive capacity of non-blotting membrane to oxyphorase.
Embodiment 4:
One, the processing of carrier-sheet glass matrix:
The processing of carrier-slide matrix is identical with embodiment 1.
Two, the preparation of blotting membrane:
Preparing method's step of blotting membrane and other material are with embodiment 1, and different is to adopt the myohaemoglobin of 0.084 μ mol/L as template molecule.
Three, the wash-out of template molecule:
The elution step of template molecule is identical with embodiment 2.
Four, molecular engram film is to the absorption of target protein:
Molecular engram film is identical with embodiment 1 to the adsorption step of target protein, and experimental result shows that molecular engram film is higher than the adsorptive capacity of non-blotting membrane to oxyphorase to the adsorptive capacity of myohaemoglobin.
Claims (7)
1. the preparation method of a protein molecule engram film the steps include:
(1), the pre-treatment of the carrier sheet glass of preparation protein molecule engram film:
Deckglass is put into beaker, the clean-out system that adds 1～5 gram in 300～500mL deionized water, ultrasonic 5～20min; Another beaker is put in the deckglass taking-up, after 3～5 flushings of deionized water, in the 1mol/L sodium hydroxide solution, soaked and ultrasonic 5～20min; Take out deckglass, to remove the sodium hydroxide solution that remains on the slide, after 3～5 flushings, add the ultrasonic 5～20min of deionized water with deionized water rinsing; Ionized water is poured out, added 1mol/L HCl solution soaking, ultrasonic 5～20min; Take out deckglass, to remove the HCl solution that remains on the deckglass, after 3～5 flushings, add the ultrasonic 5～20min of deionized water with deionized water rinsing; At last above-mentioned deckglass is taken out, dry, subsequent use;
(2), the preparation of template albumen, function monomer and cross-linking monomer solution:
With phosphate buffered saline buffer compound concentration 10
-6～10
-8The BHb solution of M takes by weighing and reaches 400: 1 water-solubility function monomers with the proteic mole ratio of template and be added in the 1mL protein solution mixing; 4 ℃ of placements are spent the night in refrigerator, add N again, N '-methylene-bisacrylamide; Ultrasonic degas 5～10min behind logical nitrogen 5～10min, adds initiator 5～10 μ L successively; And the mol ratio of function monomer between 500～1000 with catalyzer 1 μ L, mixing;
Described water-solubility function monomer is acrylic amide, USAF RH-1, acrylic or methacrylic acid;
(3), between two slides, carry out polyreaction:
Behind the protein template molecule in the step (2), function monomer, cross-linking monomer and initiator mixing; Drawing 30～50 μ L with pipettor slowly drips on slide glass; Covered is covered drop entirely, polymerization 2-3 hour; Open deckglass after the film forming, obtain the polymeric film the same with the deckglass area;
(4), wash-out template protein molecular:
Molecular engram film is put into beaker; The each 2mL of absorption elutriant adds wherein; Shake and survey its ultraviolet-visible light absorption value after 3-4 hour, till detecting, rinse out the eluent that remains on the film with deionized water at last less than absorption peak; Through surveying proteic content in the elutriant, judge the elution efficiency of elutriant;
(5), prepare non-blotting membrane, in the process of preparation polymeric film, do not add the template protein molecular by (1), (2), (3), (4) step;
(6), molecular engram film is to the method for calculation of the adsorptive capacity of target protein:
Molecular engram film MIPM and the non-molecular engram film NIPM that will pass through elution process put into respectively and are added with 6 orifice plates that heavily combine the template protein solution; Whenever utilize ultraviolet/visible spectrophotometer to measure the absorbance that heavily combines the template protein solution in 6 orifice plates at a distance from 15～60min; Calculate molecular engram film and non-molecular engram film to proteic adsorptive capacity according to the adsorptive capacity formula, adsorptive capacity: Q
Inhale=(C
O-C
Surplus) * V/S, wherein Q
InhaleBe MIPM and the proteic adsorptive capacity of NIPM absorption template; C
OAttach most importance to and combine the concentration of template protein solution; C
SurplusFor combining heavily to combine in 6 orifice plates after 2 hours the concentration of template protein solution;
(7), the selective adsorption evaluation method of molecular engram film:
MIPM and NIPM put into respectively fill the different sorts protein solution respectively: HEL, bovine serum albumin, myohaemoglobin; In 6 orifice plates of BHb; Leave standstill and place after 4 hours, utilize ultraviolet/visible spectrophotometer to measure the concentration of the heavy conjugated protein solution of different sorts in 6 orifice plates, calculate adsorptive capacity; Estimate blotting membrane to the proteic recognition performance of template, α value=Q through the value of trace factor-alpha
MIPM inhales/ Q
NIPM inhales, in the formula: Q
MIPM inhalesBe the loading capacity of blotting membrane to certain protein soln; Q
NIPM inhalesBe the loading capacity of non-blotting membrane to certain protein soln.
2. the preparation method of a kind of protein molecule engram film according to claim 1, it is characterized in that: the carrier of described blotting membrane is simple glass, silica glass or mica.
3. the preparation method of a kind of protein molecule engram film according to claim 1; It is characterized in that: described template albumen be water-soluble and with function monomer, the compatible albumen of cross-linking monomer, be selected from oxyphorase, myohaemoglobin, the bovine serum albumin any one.
4. the preparation method of a kind of protein molecule engram film according to claim 1; It is characterized in that: described is water-solubility function monomer and cross-linking monomer; Can not make template albumen generation sex change; Be selected from water-solubility function monomer acrylic amide or USAF RH-1 or acrylic or methacrylic acid a kind of or two to four kinds and cross-linking monomer N wherein, N '-methylene-bisacrylamide.
5. the preparation method of a kind of protein molecule engram film according to claim 1; It is characterized in that: described elutriant is buffered soln, salts solution, alkaline solution and the denaturing agent of template albumen wash-out from the blotting membrane, wherein a kind of of phosphate buffer soln or NaCl salts solution or NaOH alkaline solution or the denaturing agent sodium lauryl sulphate that is selected from various concentration or two to four kinds.
6. the preparation method of a kind of protein molecule engram film according to claim 1, it is characterized in that: described initiator is selected from thermal initiator and light trigger, is selected from wherein a kind of of Potassium Persulphate, ammonium persulphate, Thiocarb.
7. the preparation method of a kind of protein molecule engram film according to claim 1, it is characterized in that: described catalyzer is the tetramethyl-diethylamine.
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| CN107574206A (en) * | 2016-07-05 | 2018-01-12 | 陈栋梁 | A kind of preparation method of homoarginine hybrid peptide and its application in breast cancer treatment |
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