CN109991422A - The detection method of palmitoylation modification protein based on specific corrosioning anteserum - Google Patents
The detection method of palmitoylation modification protein based on specific corrosioning anteserum Download PDFInfo
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Abstract
The invention belongs to biochemical analysis fields; it is related to a kind of detection method of new palmitoylation modification protein based on specific corrosioning anteserum; the small peptide (Cys-Acp-Cys (Pal)) that palmitoylation is modified is coupled on carrier protein as antigen in this method; the general antiserum of anti-protein palmitoylation modification is prepared, and specifically analyzes the palmitoylation decorating state of protein in biological sample using the antiserum.General antiserum specificity produced by the present invention is more preferable, and potency is higher；This method can directly indicate the palmitoylation modification protein in all kinds of complex samples such as cell, body fluid or tissue samples using sero-fast specificity; false positive rate is low; in conjunction with Spectrometry; the high throughput identification analysis of palmitoylation modification protein in biological sample can be achieved, it is easy to operate.The present invention provides effective means for Protein Palmitoylation modification in high throughput detection biological sample.
Description
Technical field
The invention belongs to biochemical analysis fields, are related to a kind of inspection of palmitoylation modification protein based on specific corrosioning anteserum
Survey method.The invention particularly relates to the general antiserums for preparing the modification of specific recognition protein palmitoylation, and are applied to
In biological sample in the detection method of palmitoylation modification protein.
Background technique
Prior art discloses palmitoylation modification (palmitoylation), more precisely S- is acylated modification (S-
Acylation), be it is lipid-modified after a kind of dynamic, reversible protein translation, typically refer to the saturated fatty acid palm fibre of 16- carbon
Palmitic acid acid is under the action of S- acyltransferase by the way that on thioester bond covalent modification to protein cysteine residues, which passes through
Increase the hydrophobicity of protein, structure, assembling, maturation and its function of regulation protein (such as ion channel and kinases) are right
Cell signalling, metabolism and the generation of disease, development etc. all play an important role.The tradition point of Protein Palmitoylation modification
Analysis method is radioactivity palmitate metabolic marker method, is detected using the signal of autoradiograph, but this method operation is numerous
Trivial, radioactive isotope is harmful, processing cost is high, and sensitivity is not high, can only detect the palmitoylation modification albumen in living cells
Matter.The method of existing high throughput analysis palmitoylation protein is chemical method, and there are two main classes: one kind is with " palmitate "
Centered on, using nitrine or alkynyl palm acid-like substance metabolic marker cell protein, in conjunction with Staudinger connection or clickization
It learns and realizes qualitative (determining containing site) the even quantitative analysis of palmitoylation modification protein group, but the method cannot analyze tissue sample
And body fluid, palmitate analog is introduced in cell cultivation process, may result in unpredictable biological effect, and right
Modification protein analysis ability with different update speed is different；It is another kind of, it is to utilize half centered on " cysteine "
Mercapto ester bond between cystine and fatty acid modifying group is unstable, can generate free mercapto by neutral hydroxylamine solution selective hydrolysis
The characteristics of base, the acyl group to grow up-biotin displacement (Acyl-biotinyl exchange, ABE) and its deriving method, to the greatest extent
It manages the method to be widely used, but belongs to indirect detection method, false positive rate is higher.The palmitoylation modification for having not yet to see commercialization is general
Antibody report.In previous work, present inventor once modified small peptide Cys-Cys (Pal) with cysteine palmitoylation
[wherein: Pal is palmitoylation modification] is haptens, is used as antigen immunization experiment animal after being coupled with carrier protein such as KLH,
It obtains first palmitoylation and modifies general antiserum, and the specific detection point for palmitoylation modification protein in biological sample
Analysis.Although the studies above makes great progress, modified using the palmitoylation that the general antiserum can directly detect protein
State, but its specificity and potency need further improvement, be used for immunochemistry detection when dilution ratio it is lower (1:
20~30).
In view of the shortcomings of the prior art, the quasi- composition by improving haptens of present inventor, immunizing dose
With the means such as immune time, antigen is improved to the immune effect of experimental animal, and then is generated the higher palmitoylation of specificity and repaired
General antiserum is adornd, and establishes a kind of detection method of palmitoylation modification protein based on this specific antibody.
Summary of the invention
The purpose of the present invention is being based on the shortcomings of the prior art, one kind is provided and easily detects Protein Palmitoylation
The method of decorating state；It is especially a kind of to be existed using the specific recognition protein palmitoylation general antiserum of modification and the general antiserum
The method of extensive detection palmitoylation modification protein.
The technical scheme is that
Using the specific peptide fragment with the modification of cysteine palmitoylation as haptens, by itself and carrier protein
It is used as immunogene after KLH coupling, New Zealand White Rabbit is immunized；After optimized immunizing dose and immune time, using containing palmitoylation
The peptide fragment of modification and unmodified peptide fragment carry out identification screening, obtain the general of specific recognition protein cysteine palmitoylation modification
Antiserum；Immune front and back serum is collected, detects the antiserum specific recognition protein palmityl with ELISA and immunoblot assay
Change the ability of modification；Establish a kind of detection based on the specific corrosioning anteserum it is simple/complex biological sample in Protein Palmitoylation
The short-cut method of decorating state.
Further, the immunochemistry detection method of protein is modified based on antiserum specific recognition palmitoylation using this,
The palmitoylation decorating state of protein in full cell sample is analyzed, is high throughput analysis Different categories of samples (such as cell, tissue and body
Liquid) in palmitoylation modification protein provide new special analysis method.
Using the detection method in the present invention, the palmitoylation decorating state of qualitative and quantitative analysis important goal protein,
The palmitoylation decorating site of protein can be accurately analyzed in conjunction with mass spectral analysis.
Specifically, the detection method of the palmitoylation modification protein in the present invention based on specific corrosioning anteserum includes following
Step:
(1) it is haptens using the peptide fragment of cysteine palmitoylation modification, is used after it is coupled with carrier protein
The mode immunization experiment animal of subcutaneous multi-point injection；In the embodiment of the present invention, the saturated fatty acid palmitinic acid of 16 carbon will be contained
On sulfydryl by thioester bond covalent modification to cysteine, the small peptide Cys-Acp-Cys with palmitoylation modification is synthesized
(Pal), then with New Zealand White Rabbit is immunized after carrier protein KLH, to obtain the general antiserum of anti-palmitoylation modification；
Antiserum is collected in (2) the 6th immune rear neck artery bloodletting, detects sero-fast potency with elisa technique；
(3) immunochemistry inspection is carried out using haptens Cys-Acp-Cys (Pal) of the antiserum collected to various concentration
It surveys, investigates the sero-fast detection sensitivity；
(4) using antiserum obtained to the palmitoylation in liver cancer cell lines 97L and LM3 with Metastatic potential
It modifies protein and carries out immunochemistry detection, by comparing the immunochemistry signal detected in different cell lines, it is anti-to evaluate this
The specificity and validity of serum；
(5) using based on important in sero-fast immunochemistry detection method quantitative analysis cell system 97L and LM3 obtained
The palmitoylation decorating state of Protein L amin A, and high-resolution mass spectrometric analysis method is combined, identify the palm fibre of Lamin A
Palmitic acid is acylated decorating site, the feasibility of evaluation method.
It is that [wherein: Acp is aminocaproic acid to haptens, and Pal is that the present invention provides one kind based on Cys-Acp-Cys (Pal)
Palmitoylation modification] it prepares the general antiserum of specific recognition protein palmitoylation modification and is examined using the general antiserum high throughput
The method of palmitoylation modification protein, shows through experiment in survey biological sample, and general antiserum energy obtained is special in the present invention
The palmitoylation of identification of protein is modified, and has good immunochemistry detection effect to palmitoylation modification proteins/peptides section,
It can be used for the high-throughput detection of palmitoylation modification protein in simple and complex biological sample.
It advantages of the present invention and has the beneficial effect that
The peptide fragment Cys- that the method for the present invention is modified using the cysteine palmitoylation with aminocaproic acid linking arm for the first time
Acp-Cys (Pal) [wherein: Acp is aminocaproic acid, and Pal is palmitoylation modification] is haptens, is prepared for the palm fibre of high specificity
Palmitic acid is acylated to modify general antiserum, and is detected using the general antiserum to Protein Palmitoylation decorating state.With it is existing
Analysis method is compared, and this law can be used to directly indicate the palmitoylation modification egg in Different categories of samples (tissue, cell and body fluid etc.)
White matter, specificity is good, and false positive rate is low, combines with Spectrometry, can analyze palmityl in biological sample with high throughput
Change the protein of modification, strong operability is simple and easy to do.With inventor in previous work with Cys-Cys (Pal) be haptens
The palmitoylation being prepared is modified general antiserum and is compared, in antiserum preparation method of the present invention, on the one hand, with
Cys-Acp-Cys (Pal) is haptens, improves the structure of haptens, due to the introducing of linking arm aminocaproic acid, makes antigen table
Position is more prominent；On the other hand, due to the increase of immunizing dose and immune time, it is unstable thioester bond has been largely overcoming
And the problems such as water solubility due to caused by palmitinic acid strong-hydrophobicity and poor immunogenicity bring it is insufficient, made in the present invention
Standby obtained general antiserum has preferably specificity and detection sensitivity.
Detailed description of the invention
Fig. 1 is tested using dot blot investigates sero-fast specificity and detection sensitivity, in figure, Cys-Acp-Cys
It (Pal) is modification peptide fragment, Cys-Acp-Cys is non-modified peptide fragment, wherein Acp is aminocaproic acid, and Pal is palmitoylation modification
Group illustrates that the general antiserum of anti-palmitoylation modification is effective and detection sensitivity with higher.
Fig. 2 utilizes liver cancer cells 97L and the LM3 sample with Metastatic potential to evaluate sero-fast detection effect: (A)
It is antiserum made from haptens with Cys-Cys (Pal)；It (B) is antiserum made from haptens with Cys-Acp-Cys (Pal).
Illustrate horizontal with different palmitoylation modifications in different cell 97L and LM3；For same cell sample, two kinds are utilized
The pattern that antiserum obtains after being immunoreacted is similar, but there are certain specificity；And with Cys-Acp-Cys (Pal)
There is better detection effect, lower detection sensitivity and higher dilution ratio for antiserum made from haptens.
Palmitoylation decorating state of the Fig. 3 using Lamin A in antiserum western blot method detection 97L and LM3, figure
In, IP:LMNA is indicated first with the monoclonal antibody of anti-Lamin A by the Lamin A co-immunoprecipitation in 97L and LM3 cell, then
By Lamin A equivalent loading, the anti-Lamin A antibody of the antiserum of the invention obtained and commercialization is utilized respectively to its palmityl
Change modification level and expressing quantity is detected, illustrates the palm that the antiserum can be used for quantitative detecting analysis protein
Acylated modification is horizontal.
Fig. 4 is verified that the method for the present invention detects as a result, in figure by existing ABE method, and "-" and "+" respectively indicate " no
Add " and " adding " azanol and TS-6B enrichment handle；Diagram shows the result detected using existing general ABE method and this hair
The result that bright method detects is consistent, and the palmitoylation modification of lamin A is horizontal in 97L cell is higher than in LM3, illustrates this
Antiserum is effective.
The peptide fragment AQNTWGCGNSLR mass spectrum tandem figure of Fig. 5 Protein L amin A, illustrates Lamin A and palm fibre has occurred
The acylated modification of palmitic acid, conclusion is consistent with the result that the method for the present invention detects, and illustrates that the antiserum is effective.
Specific embodiment
The present invention is further elaborated below with reference to chart and specific embodiment, so that those skilled in the art can be more
Clearly learn technical solution of the present invention, not limitation of the present invention.
Embodiment 1 prepares immunizing antigen
It adopts and synthesizes the peptide fragment containing palmitoylation modification and its control peptide fragment with method known in this field, and palm will be contained
The peptide fragment and carrier protein couplet of acylated modification in the present embodiment, complete the peptide by Shanghai Rui Xing Bioisystech Co., Ltd
Duan Hecheng and albumen coupling: the small peptide of synthesis the small peptide Cys-Acp-Cys without palmitoylation modification and the modification containing palmitoylation
Cys-Acp-Cys (Pal), and Cys-Acp-Cys (Pal) is coupled on carrier protein KLH, it is expressed as KLH-Cys-Acp-
Cys(Pal)。
2 antigen of embodiment is immune, prepares palmitoylation and modifies general antiserum
It regard KLH-Cys-Acp-Cys (Pal) obtained in embodiment 1 as antigen, New Zealand White Rabbit is immunized in six times,
In, the immunizing dose of first time is 200 μ g/ rabbits, and subsequent each immunizing dose is 100 μ g/ rabbits, after six times are immune, conventional neck
Arterial blood letting collects antiserum and is stored in -20 DEG C.
Embodiment 3 investigates sero-fast specificity using competitive ELISA
ELISA Plate is added as antigen in Cys-Acp-Cys (Pal) (100 μ g/ml), 4 DEG C of coatings are stayed overnight, using this field
Well known ELISA method is detected, by the antiserum institute for detecting different dilution ratios (start at 1: 100,1: 3 gradient dilution)
The immune signal of generation evaluates the sero-fast optimal use dilution ratio obtained in embodiment 2；By comparing immune front and back blood
Immune signal caused by clear, and carried out using modification peptide fragment Cys-Acp-Cys (Pal) and non-modified peptide fragment Cys-Acp-Cys
Competitive assay investigates the sero-fast validity and specificity；The results are shown in Table 1, be immunized before serum to modification peptide fragment and
Non-modified peptide fragment does not respond to, and the antiserum after being immunized has very strong immune response, and competitive assay to modification peptide fragment
The results show that the antiserum for palmitoylation modification have well selectivity, although also having centainly to non-modified peptide fragment
Response, and the sero-fast best dilution ratio be~1: 900.
Table 1. investigates sero-fast validity using ELISA method
Illustrate in table: the 1st is classified as sero-fast dilution ratio, and wherein BLANK is the preimmune serum of the New Zealand White Rabbit,
As a control group；2nd~4 column are to modify peptide fragment as antigen coat ELISA Plate, wherein the 2nd column are indicated antiserum by specific
It being immunoreacted after dilution proportion, the 3rd column indicate after reacting modification peptide fragment in advance with the antiserum of specific dilution ratio, then
It is added into ELISA Plate and is immunoreacted, the 4th column indicate that non-modified peptide fragment and the antiserum of specific dilution ratio is preparatory
After reaction, then it is added into ELISA Plate and is immunoreacted；5th column, using non-modified peptide fragment as antigen coat ELISA Plate after use
Antiserum is immunoreacted.Used modification peptide fragment and non-modified peptide fragment concentration are 100 μ g/ml.
Embodiment 4 carries out the sero-fast detection sensitivity of dot blot experimental identification using antiserum
It is with the modification peptide fragment Cys-Acp-Cys (Pal) (0,0.625,1.25,2.5,5,10fmol/ μ l) of various concentration
Sample (applied sample amount is 1 μ l), after the antiserum obtained in embodiment 2 is diluted with 1: 1000, using dot well known in the art
Blot method is detected；The result shows that the antiserum has very strong immune letter to modification peptide fragment Cys-Acp-Cys (Pal)
Number, under the same terms, it is nearly no detectable the signal of non-modified peptide fragment；Moreover, modification peptide fragment of the antiserum to extremely low concentration
(such as 0.625fmol) still has good response signal, illustrates that the antiserum has inspection well to palmitoylation modification peptide fragment
Survey specificity and sensitivity.
Embodiment 5 using antiserum detection there is palmitoylation in the liver cancer cells 97L and LM3 of Metastatic potential to modify
Protein
Further to investigate the sero-fast effect obtained in embodiment 2, used in the present embodiment well known in the art
Western blot method detects palmitoylation modification protein in liver cancer cell lines 97L and LM3, while relatively more real
Apply the antiserum obtained in example 2 and in the prior art with Cys-Cys (Pal) for sero-fast detection effect made from haptens.
Preprocess method before antiserum use:
(1) be antiserum made from haptens with Cys-Cys (Pal): the antiserum after collection need to use ammonium sulfate precipitation
Method pretreatment, it may be assumed that antiserum is centrifuged 30min under the conditions of 20000 × g, 4 DEG C, collects supernatant；Take 400 μ l supernatants and isometric
After physiological saline mixing, 400 μ l saturated ammonium sulfates are slowly added dropwise under stirring condition, 4 DEG C of precipitates overnights precipitate albumen sufficiently；
It is centrifuged 10min under the conditions of 20000 × g, 4 DEG C, abandons supernatant；With 240 μ l physiological saline solution protein precipitations, 160 μ l are added dropwise
Saturated ammonium sulfate precipitates 1hr under the conditions of 4 DEG C；It is centrifuged 10min under the conditions of 20000 × g, 4 DEG C, is discarded supernatant；240 μ l are added
Physiological saline solution precipitating, and ultrafiltration removes excessive salt, is collected as 100 μ l solution, it is spare；To be used after 1: 20 dilution.
(2) antiserum obtained in embodiment 2: directly to be used after 1: 1000 dilution,
The concrete analysis step of palmitoylation modification level includes: in cell sample
1) method culture liver cancer cell lines 97L and LM3 well known in the art are pressed；
2) cell protein extracts: collecting 97L and LM3 cell, is separately added into lysate (25mM HEPES, 25mM
NaCl, 1mM EDTA, 1 × protease inhibitor cocktail (EDTA Free, Roche Diagnostics) and 1
× PMSF, pH 7.4), 4 DEG C of ultrasound cracking carry out Protein Extraction, and in 4 DEG C 20,000 × g is centrifuged resulting protein cleavage product
After 30min, supernatant is taken to carry out quantification of protein according to BCA method well known in the art using ultraviolet-visible spectrophotometry；
3) immunoblot experiment: taking the 100 above-mentioned protein examples of μ g, and three (2- carboxyethyl) phosphines of final concentration of 20mM are added
(TCEP) disulfide bond in protein is opened in reduction, and the n-ethylmaleimide (NEM) that final concentration of 50mM is added is protected from light
It reacts, the free sulfhydryl group in closed protein matter；12% polyacrylamide gel electrophoresis is carried out, is existed by method well known in the art
The protein band in gel is transferred on pvdf membrane (Millipore company) in Bio-Rad electrotransfer system, film is placed in
It is stayed overnight for 4 DEG C in TBS-T confining liquid containing 5% skimmed milk power.It is separately added into the above two general antiserum pre-processed and shakes 4 DEG C
It is incubated overnight, after washing film with TBS-T, 1: 8000 diluted goat-anti rabbit secondary antibody (Thermo Scientific), room temperature concussion is added
It is incubated for 1 hour, TBST washes film again, the super quick developing solution (Thermo Scientific) of ECL is added, with ImageQuant ECL
Instrument (GE company) capture colour developing image.As shown in Fig. 2, cell line 97L and LM3 with Metastatic potential, palmityl
Changing modification level has apparent difference, and the modification level of protein is apparently higher than LM3 cell in 97L；For same cell sample
Product, the pattern obtained after being immunoreacted using two kinds of antiserums is similar, but due to antigen difference, two kinds of antiserum inspections
Surveying obtained immune signal, there are certain specificity；And it is more using band caused by the antiserum obtained in embodiment 2
In being antiserum made from haptens with Cys-Cys (Pal), the results show that antiserum produced by the present invention repairs palmitoylation
Decorations have better detection effect, lower detection sensitivity and higher dilution ratio.
Embodiment 6 utilizes the palmitoylation of Protein L amin A in antiserum quantitative analysis liver cancer cell lines 97L and LM3
Modification is horizontal
(1) 97L and LM3 cell culture is carried out by embodiment 5, by co-immunoprecipitation method well known in the art, with anti-
The monoclonal antibody of Lamin A Immunological purification Lamin A albumen from equivalent 97L and LM3 cell；The Lamin that purifying is obtained
It is general anti-using what is obtained in embodiment 2 after A protein sample carries out gel electrophoresis separation, transferring film using method described in embodiment 5
The palmitoylation that serum western blot detects Lamin A is modified horizontal；
If Fig. 3 shows, for the Lamin A albumen of identical applied sample amount, after being incubated for using the antiserum, in 97L cell
Lamin A has apparent immune signal, illustrates that palmitoylation modification has occurred in Lamin A, and almost examines in LM3 cell protein
The immune signal for not detecting it illustrates that the palmitoylation modification level of the Lamin A in the cell of Metastatic potential is different
, and the antiserum can be used for the palmitoylation modification level of quantitative detecting analysis protein；
(2) the palmitoylation decorating state of Lamin A albumen is verified using existing ABE method:
97L and LM3 cell protein obtained in embodiment 5 (2) is subjected to reduction alkane by method shown in embodiment 5 (3)
After baseization processing, after removing wherein excessive TCEP and NEM using methanol-chloroform protein precipitation quality sample, with containing 1%SDS's
After 50mM Tris-HCl (pH 7.4) weight is molten, 97L the and LM3 protein example of equivalent is taken to carry out subsequent processing；It respectively will be above-mentioned
Protein example is divided into two parts, and azanol processing is not added in portion, and (purpose is to detect the protein expression water of Lamin A in 97L and LM3
It is flat)；Another hydroxylamine solution room temperature that pH 7.4 is added handles 2~3hr, with the solid support material Thiopropyl of commercialization
By product description operation progress selective enrichment, (purpose is had occurred in detection 97L and LM3 to Sepharose 6B (TS-6B)
The Lamin A protein level of palmitoylation modification)；Final concentration 10mM dithiothreitol (DTT) solution is added by target protein from TS-
It is dissociated on 6B material, then carries out SDS-PAGE separation, be incubated for the monoclonal antibody of anti-Lamin A and carry out western blot
Analysis；Since palmitoylation modification is palmitinic acid by the modification to protein cysteine residues of mercapto ester bond, and mercapto ester bond
It is unstable, it can be hydrolyzed in neutral hydroxylamine solution, free sulfhydryl group is generated at decorating site, so as to utilize energy and sulfydryl
The solid support material of idiosyncrasy such as TS-6B realizes selective enrichment to it；If Fig. 4 shows, Lamin A albumen is hydrolyzed through azanol
After can be enriched with by TS-6B, illustrate in Lamin A exist the mercapto ester bond sensitive to azanol, it is possible to have occurred palmitoylation modification,
And the Lamin A albumen that can be enriched in 97L cell is more, illustrates the palmitoylation modification water of Lamin A in 97L cell
It puts down than LM3 high.This is consistent with result obtained in embodiment 6 (1)；
(3) the 97L cell holoprotein sample for taking 100 μ g, is added final concentration of 20mM TCEP solution in 56 DEG C of oscillating reactions
1hr is to open the disulfide bond in protein；Final concentration of 50mM NEM is added and is protected from light 1hr closing free sulfhydryl group；Using first
After alcohol-chloroform protein precipitation quality sample removes wherein excessive TCEP and NEM, with the 50mM Tris-HCl (pH containing 1%SDS
7.4) after weight is molten, the hydroxylamine solution room temperature that pH 7.4 is added into protein example handles 2~3hr, is carried with the solid phase of commercialization
Body material TS-6B carries out selective enrichment by product description operation；Final concentration 10mM dithiothreitol (DTT) solution is added by target
After protein is dissociated from TS-6B material, final concentration of 100mM iodoacetamide solution room temperature is added, be protected from light under the conditions of react
45min is alkylated reaction；Sequencing grade trypsase (Promega) is added by 1: 50 (enzyme: protein), by known in this field
Protein digestion method in 37 DEG C enzymatic hydrolysis overnight after, by C18 column desalination well known in the art, vacuum freeze-drying concentration, with 0.1%
FA aqueous solution is resuspended, and 15 μ l peptide fragment mixtures is taken to carry out LC-MS separation analysis, using Acclaim PepMap C18 column, 75 μm
× 50cm (Thermo Scientific company) carries out chromatographic isolation, chromatography gradient: B phase (ACN-0.1%FA) is in 120min ladder
From 2% linear rise to 45% in degree, flow velocity 300nL/min；Using Q Exactive mass spectrograph (Thermo
Scientific company) be analyzed by mass spectrometry, mass spectral analysis the result shows that, the peptide fragment AQNTWGCGNSLR of Lamin A albumen
In cysteine residues have occurred palmitoylation modification, it is consistent with the conclusion that embodiment 6 (1) obtains if Fig. 5 shows.
Claims (4)
1. a kind of detection method of the palmitoylation modification protein based on specific corrosioning anteserum, which is characterized in that known using special
Other palmitoylation modifies the palmitoylation decorating state of general antiserum detection protein comprising step:
(1) using the small peptide (Cys-Acp-Cys (Pal)) that palmitoylation is modified as haptens, it is even with carrier protein KLH
Join immunization experiment animal, obtains the general antiserum of anti-palmitoylation modification；In the Cys-Acp-Cys (Pal): Acp is ammonia
Base caproic acid, Pal are palmitoylation modification；
(2) antiserum is collected after being immunized, and detects sero-fast potency with elisa technique；
(3) immunochemistry detection is carried out by haptens small peptide of the different dilution ratios to various concentration with the antiserum collected,
Investigate sero-fast detection sensitivity and dilution ratio；
(4) Protein Palmitoylation decorating state and its level in different cells are detected with the antiserum immunochemistry collected
Variation；
(5) high-resolution mass spectrometry method is combined, the Protein L amin A of the western blot testing result positive is identified
Analysis, the feasibility of evaluation method.
2. method according to claim 1, which is characterized in that in the step (1), palmitinic acid is covalently repaired by thioester bond
It adorns on the sulfydryl of cysteine, with immunization experiment animal after carrier protein KLH coupling；Pass through drawing for linking arm aminocaproic acid
Enter, keep epitope more prominent, to improve immune effect.
3. method according to claim 1, which is characterized in that the antiserum in the step (2) is received after being immune the 6th time
Collection.
4. method according to claim 1, which is characterized in that the cell in the step (4) is with Metastatic potential
Cell 97L and LM3.
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