Method for preparing N-terminal Gly2 myristoylated ARF1 antibody for immunohistochemistry experiments

Abstract

This invention provides a method for preparing N-terminal Gly2-myristylated ARF1 antibody for immunohistochemical experiments. The method involves immunizing animals with a polypeptide whose amino acid sequence is shown in SEQ ID NO.2 to obtain the ARF1 antibody. The ARF1 antibody prepared by this method can be used in immunohistochemical experiments.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing second-position N-myristylated ARF1 antibodies for immunohistochemistry and immunofluorescence experiments. Background Technology

[0002] ADP-ribosylation factor 1 (ARF1) is a core small GTPase regulating intracellular membrane transport, vesicle transport, and organelle homeostasis in eukaryotic cells. The myristoylation modification of the second glycine residue (Gly2) at the N-terminus is irreplaceable for the normal activation and function of ARF1. This post-translational modification is a prerequisite for ARF1 to perform its biological functions. Without specific myristoylation at the second Gly2 site, ARF1 cannot complete conformational switching and membrane localization, and will completely lose its intracellular physiological activity.

[0003] Furthermore, studies have shown that N-terminal Gly2 myristoylation modification is not only essential for ARF1 to maintain basic cellular function, but also widely involved in multiple physiological and pathological processes such as immune response, pathogen infection, and tumor progression. Normally myristoylated ARF1 can regulate the secretion of immune-related factors and the activation of signaling pathways, helping the host to complete immune defense; while various pathogens can inhibit the function of ARF1 by specifically cleaving the myristoyyl chain, thus achieving immune escape. Simultaneously, abnormally upregulated myristoylation levels of ARF1 in tumor cells significantly enhance cellular secretion, migration, and anti-apoptotic capabilities, making this modification site an important potential target for the development of antitumor and antiviral drugs.

[0004] In summary, N-terminal Gly2 myristoylation is a core and essential modification for ARF1, encompassing its molecular conformational regulation, membrane localization, activity cycling, and downstream physiological functions, with significance throughout the entire process of ARF1 action. It is not only a core molecular mechanism for maintaining the homeostasis of the eukaryotic cell endometrial system and ensuring the transport of essential substances, but it is also closely related to the occurrence and development of various diseases. A deeper understanding of the mechanism of action of this site modification can not only improve the theoretical framework of endometrial transport regulation but also provide important theoretical support for targeted therapy of related diseases.

[0005] However, there are currently no successfully developed antibodies on the market that can be used for ARF1 N-terminal Gly2 cardiomylation specific antibody. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for preparing N-terminal Gly2-myristoylated ARF1 antibody for immunohistochemistry and immunofluorescence experiments, which solves the problem of analyzing N-terminal Gly2-myristoylated ARF1 in mouse samples by immunohistochemistry and immunofluorescence.

[0007] One aspect of the present invention provides a polypeptide for preparing an ARF1 antibody, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Another aspect of the present invention provides a gene fragment capable of encoding the aforementioned polypeptide.

[0009] Another aspect of the present invention provides the use of the above-described polypeptide in the preparation of ARF1 antibodies.

[0010] Furthermore, the polypeptide is used to prepare ARF1 antibody after conjugation.

[0011] Furthermore, the polypeptide can be detected by immunohistochemistry and / or immunofluorescence assays.

[0012] Another aspect of the present invention provides a method for preparing ARF1 antibodies using the above-mentioned polypeptide, wherein the method involves immunizing an animal with the polypeptide to induce the production of ARF1 antibodies, and then collecting the animal serum to obtain ARF1-containing antibodies.

[0013] Furthermore, the animal in question is either a rabbit or a sheep.

[0014] Furthermore, the polypeptide is used to immunize animals after conjugation. The conjugation method is a prior art technique, and specific details can be found in the embodiments described in the specification.

[0015] Furthermore, the immunized animals include those receiving primary immunization and those receiving booster immunization.

[0016] Furthermore, the method also includes extracting and purifying animal serum. The extraction and purification can be performed using chromatography, such as affinity purification of animal serum using a peptide cross-linking chromatography column. Peptide cross-linking chromatography columns can be purchased or prepared in-house; specific preparation methods can be found in the examples section of this specification.

[0017] As described above, the method for preparing the second-position glycine N-myristylated ARF1 antibody of the present invention has the following beneficial effects: The antibody prepared using the method described in this paper can produce a highly efficient N-myristoylated ARF1 (Gly2) antibody. This antibody can effectively detect N-terminal Gly2-myristoylated ARF1, which is of great significance for cancer progression and prognosis. Attached Figure Description

[0018] Figure 1 The diagram shows an immunohistochemical assay of N-myristoylated ARF1 (Gly2) antibody. Detailed Implementation

[0019] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0020] Example 1: Determination and optimization of antigen amino acid sequence Based on the results of antigenic epitope analysis using Abdesigner software, and considering factors such as hydrophilicity, antigenicity, surface feasibility, and ease of synthesis, as well as the in vivo natural cleavage characteristics of ARF1, the initial methionine was removed, and the germination-modified ARF1 antigenic polypeptide sequence was finally determined to be GNIFANLF (SEQ ID NO.2). This polypeptide is used for carrier protein cross-linking and subsequent affinity purification, providing an immunogen for the preparation of specific antibodies.

[0021] Example 2: Peptide Conjugation 1. KLH dissolution: Dissolve KLH in degassed pure water according to the proportions in the table below.

[0022] Table 1 KLH <![CDATA[H2O]]> 100 mg 12.5ml 2. Add SMCC: Weigh out Sulfo-SMCC according to the proportions in the table below and add it to KLH solution. (Note: The solubility of Sulfo-SMCC in water or other commonly used buffer solutions is approximately 10 mM, and its solubility decreases with increasing salt concentration.) Table 2 KLH SMCC 100 mg 25mg 3. Reaction: The KLH-Sulfo-SMCC solution was slowly stirred at room temperature for 60 minutes.

[0023] 4. Desalting: The KLH-Sulfo-SMCC solution is used to remove free Sulfo-SMCC using a desalting column. The desalting column is equilibrated with 3 column volumes of cross-linking buffer, and then the sample is loaded. If the volume is large, it can be loaded in several batches. After the KLH-SMCC solution enters the gel bed, it is eluted with cross-linking buffer. The protein peak containing KLH-SMCC is collected by measuring the absorbance at 280 nm. Each peak collection is combined. (Note: The cross-linking agent can react with the desalting column, so this desalting column can only be used to remove free SMCC.) 5. Determination: The concentration of KLH-SMCC was determined using the Lowry method.

[0024] 6. Dispensing: Dispense KLH-SMCC into 3 mg / tubes according to the measured protein concentration. Weigh the required peptide (peptide to KLH weight ratio is 1:1).

[0025] 7. Dissolve the polypeptide in sterile, deaerated purified water to a final concentration of 20 mg / ml. If complete dissolution is difficult after dissolution, a dissolving aid can be used.

[0026] 8. Mix KLH-SMCC with the peptide solution.

[0027] 9. React the mixture overnight at 4°C in a silent mixer.

[0028] 10. Weigh out L-cysteine ​​for blocking, with a cysteine ​​to KLH weight ratio of 2:1.

[0029] 11. Dissolve cysteine ​​in an appropriate amount of water, and then add the dissolved cysteine ​​solution to the polypeptide-KLH mixture.

[0030] 12. Gently shake the mixture at 4°C for 2 hours.

[0031] 13. After sealing, transfer the mixture to a newly prepared dialysis bag and dialyze with 0.01M PBS. Change the dialysate at least twice during the process, with an interval of at least 4 hours between each dialysate.

[0032] 14. After dialysis, collect all contents of the dialysis bag, especially any precipitates that may have formed during dialysis (which are usually the best immunogens), and rinse the dialysis bag with 0.01M PBS and collect the rinse solution.

[0033] 15. Based on experience, the theoretical conjugation rate of peptides to KLH is approximately 30%, and this value applies to most peptides. For example, conjugating 10 mg of KLH with 10 mg of peptide will theoretically yield 13 mg of peptide-KLH conjugate. Based on the theoretical yield, dilute the peptide-KLH conjugate to 1 mg / ml.

[0034] 16. Dispensing: Dispense into 300ug and 200ug portions, label them, and place them in sample boxes.

[0035] Example 3: Immunizing animals with antigens 1. Antigen emulsification 1.1 Before immunization, according to the plan, prepare the dual syringes required for antigen emulsification and the glass syringes for immunization in advance, and affix labels.

[0036] 1.2 On the day of immunization, according to the plan, take out the peptide-KLH antigen from the -70℃ freezer and prepare Freund's adjuvant according to the immunization type. Use complete Freund's adjuvant for basic immunization and incomplete Freund's adjuvant for booster immunization. This step must be completed by two people and checked one by one.

[0037] 1.3 Dilute the antigen to 1 ml / sheep with 0.01 M PBS.

[0038] 1.4 Draw all the antigen into one emulsification syringe, and draw an equal volume of adjuvant into another syringe, ensuring all air is expelled from the syringes. Before drawing in the liquid, a small amount of paraffin oil can be applied to the inner handle of the syringe to reduce resistance during emulsification.

[0039] 1.5 Connect the two syringes with a double needle and tighten the needle with tweezers or hemostats to prevent leakage.

[0040] 1.6 Forcefully and quickly push the antigen solution into the adjuvant solution (do not push the adjuvant into the antigen solution first), then repeatedly push and pull the dual syringe until emulsification is complete. Apply moderate force when pushing and pulling to avoid the dual syringe needle breaking open.

[0041] 1.7 Fill a clean petri dish with cold water, and drop a small drop of the emulsified antigen-adjuvant mixture into the water. If the drop does not spread, the emulsification is successful. If it spreads, continue pushing and pulling to emulsify until the emulsification is successful.

[0042] 1.8 Transfer the emulsified antigen, which has passed inspection, into a glass syringe for injection using a double needle, and attach the needle for use. Before transfer, verify that the labels on the emulsifying syringe, the syringe for injection, and the needle are consistent.

[0043] 2. Basic Immunization 2.1 Remove the sheep requiring immunization from the cage and place them on the operating table. Handle the sheep according to the prescribed method, using gentle movements. If necessary, gently stroke the sheep until it calms down before handling it or performing other operations, minimizing the risk of startling or mechanical injury. Always verify the sheep's ear tag before handling.

[0044] 2.2 One person holds the sheep still while another person uses curved surgical shears to cut the wool from front to back on the left side of the back, with a width of 1-2 cm.

[0045] 2.3 Disinfect the skin at the haircutting site with a cotton ball soaked in 75% alcohol.

[0046] 2.4 Verify that the syringe label number matches the sheep's ear number, and then begin immunization. For basic immunization, administer subcutaneous injections at 8-10 sites, with 0.3-0.5 ml of antigen at each site.

[0047] 3. Boost immunity 3.1 Perform booster immunizations according to the company's plan. The basic procedures and precautions for booster immunizations are the same as for basic immunizations.

[0048] 3.2 The first booster immunization should be performed by shearing the sheep on the right side of its back. It is important to avoid injecting the antigen into areas with ulcers or granulomas from the primary immunization. This should also be observed during subsequent booster immunizations.

[0049] 3.3 Enhanced Immunization: In addition to the five subcutaneous immunization sites on the back, two more intramuscular injection sites were added. The skin on the inner thighs of the sheep was disinfected with alcohol swabs, then the needle was inserted into the muscle, injecting 0.1–0.3 ml of antigen at each site.

[0050] Example 4 Serum Collection 1. Blood samples are generally collected twice: blood sample A is collected seven days after the first booster immunization, and blood sample B is collected seven days after the second booster immunization. A positive blood sample is collected seven days after the third booster immunization and retained as the test serum.

[0051] 2. Before blood collection, blood collection tubes should be prepared in advance according to the plan and clearly marked. This step must be checked by someone.

[0052] 3. Serum testing: one tube per sheep, according to sheep number.

[0053] 4. Remove the sheep to be blooded from the cage and secure it to the sheep restraint. Handle the sheep gently, following the prescribed method. If necessary, gently stroke the sheep until it calms down before handling it or performing other operations. Minimize the risk of startling the sheep or causing mechanical injury. Always verify that the blood collection tube number and the sheep's ear number match when handling the sheep.

[0054] 5. Trim the hair from the sheep's ear edges, wipe the auricle with an alcohol swab, and gently tap the vein area with your fingernail to make the veins congested and prominent.

[0055] 6. Puncture the vein with a disposable syringe needle sterilized with 75% ethanol. After bleeding, collect whole blood in a 0.5ml centrifuge tube. Collect approximately 0.3-0.4ml of whole blood from each sheep.

[0056] 7. After blood is drawn, disinfect the wound with an alcohol swab.

[0057] 8. Return the sheep to their respective cages.

[0058] 9. Let the collected whole blood stand at room temperature for about 1 hour to allow the serum to separate out.

[0059] 10. The whole blood was centrifuged using a palm-sized centrifuge to separate the serum. During centrifugation, attention should be paid to the balance of the centrifuge tubes.

[0060] 11. Use a pipette to aspirate the serum and transfer it into a sterile 0.5ml centrifuge tube.

[0061] Example 5 Preparation of affinity chromatography column 1. Conjugation of peptides with Sulfolink Gel 1.1 Remove the Sulfolink Gel from the 4°C freezer and allow it to reach room temperature. Place the column vertically and open the top cap. Wash the column with one column volume of crosslinking buffer, leaving a small amount of crosslinking buffer at the bottom of the column, and attach the bottom disc.

[0062] 1.2 Take a well-mixed suspension of Sulfolink Gel (the final gel bed volume is 2 ml) and load it into the chromatography column. Equilibrate with at least 20 ml of cross-linking buffer, and then attach the bottom cap of the chromatography column.

[0063] 1.3 Dilute the dissolved 2 mg peptide into 2 ml cross-linking buffer.

[0064] 1.4 Add the polypeptide solution to the chromatography column, attach the top cap to the chromatography column, and label the chromatography column.

[0065] 1.5 Shake and mix the chromatography column at 4°C and react overnight.

[0066] 1.6 The next day, take the chromatography column and let it stand at room temperature for 30 minutes.

[0067] 1.7 Remove the top and bottom caps of the chromatography column in top-to-bottom order to drain the polypeptide solution from the column and collect it into a clean tube.

[0068] 1.8 Wash the chromatography column with 20 ml of cross-linking buffer (the amount of cross-linking buffer should be no less than 5 column volumes).

[0069] 2. Block nonspecific sites on Sulfolink Gel 2.1 Reopen the bottom cover of the chromatography column.

[0070] 2.2 Add 2 ml of 50 mM L-cysteine ​​solution to the column to react with the gel, and then attach the top cap of the chromatography column (the volume ratio of L-cysteine ​​solution to gel is 1:1).

[0071] 2.3 Mix and react at room temperature for 2 hours.

[0072] 2.4 Let stand at room temperature for 30 minutes.

[0073] 2.5 Discharge excess cysteine ​​solution.

[0074] 3. Washing the column 3.1 Remove the top and bottom caps of the chromatography column in the order of top to bottom, and drain the liquid from the chromatography column.

[0075] 3.2 Wash the chromatography column with 20 ml of PBS, and attach the disc gel fixative sheet at the top during the first wash.

[0076] 3.3 Then wash the chromatography column with 20 ml of 1 M NaCl, 20 ml of PBS, and 20 ml of 100 mM Gly-HCl buffer, respectively.

[0077] 4. Storage of peptide cross-linking columns 4.1 Replace the bottom cap and add 10 ml of storage solution to the chromatography column, allowing it to flow out until the upper liquid surface is about 0.8 cm away from the disc gel fixing surface.

[0078] 4.2 Reinstall the top cover.

[0079] Example 6 Affinity Purification Material 1. Animal serum 2. Peptide cross-linking chromatography column 3. 1M Tris·HCl, pH 7.5 4. 100mM glycine buffer, pH 3.0 5. 10mM PBS pH 7.4 6. 10mM PBST pH7.4 (containing 0.05% Tween 20) 7. Storage fluid Note: All liquids entering the chromatography column must be degassed.

[0080] Operating steps 1. Serum treatment 1.1 Take out the serum samples from each batch to be processed and mix them. Control the reaction volume to be about 40 ml each time, and retain the samples for ELISA detection.

[0081] 1.2 Adjust the pH value by adding 1 / 10 volume of 1M Tris pH7.5 buffer according to the serum volume, mix well at room temperature and let stand for 20 minutes.

[0082] 1.3 Filter the pH-adjusted serum sample through a sterile 0.45µm membrane.

[0083] 2. P-peptide column chromatography 2.1 Remove the cross-linked P-peptide chromatography column from the 4°C freezer and allow it to reach room temperature. First, remove the top cap to prevent air from entering the gel bed.

[0084] 2.2 Remove the bottom cap to allow excess storage buffer to drain. Wash the column with 20 ml of 10 mM PBS (pH 7.4).

[0085] 2.3 Take 2 ml of P-peptide-conjugated Sulfolink Gel from the column and mix it with the treated serum sample. Mix the sample by rotating it on a silent rotator and react overnight at 4°C or for 2 hours at room temperature.

[0086] 2.4 Place the empty chromatography column upright on the chromatography rack. Add 10 ml of PBS and use a syringe plunger to remove air from the column film and the area beneath it. Pack the serum-Sulfolink Gel mixture into the column stepwise. Sulfolink Gel will deposit to form a gel bed (to prevent the gel bed from drying out). Wash the column with 5-7 ml of PBS (to remove serum from the gel). Collect the eluent in a clean bottle.

[0087] 2.5 Wash the chromatography column with 20 ml of 10 mM PBS pH 7.4. When the liquid level is 1-2 cm below the gel surface, plug the bottom cap of the chromatography column, attach the disc film on the gel, wash the chromatography column again with 10 ml of PBS pH 7.4, and finally wash the chromatography column with 20 ml of 10 mM PBS pH 7.4 containing 0.05% Tween 20 solution.

[0088] 2.6 For each sample, prepare two 1.5ml collection tubes containing 100ul of 1M Tris·HCl at pH 7.5 and a 30m bottle, and label them accordingly.

[0089] 2.7 First, wash the chromatography column with 1 ml of 100 mM glycine buffer (pH 3.0), without collecting the eluent. Then wash the column twice more with 1 ml of 100 mM glycine buffer (pH 3.0), collecting the eluent from each wash into a 1.5 ml centrifuge tube. Mix well to restore the pH to neutral. Depending on the amount of protein, decide whether to collect the eluent using a bottle or a cuvette. Finally, collect until no protein remains.

[0090] 2.8 Measure the colorimetric value of each collected solution at 280 nm. Combine the solutions with an absorbance greater than 0.1 into the elution buffer for further purification.

[0091] 2.9 The serum eluent after P-peptide chromatography can be reabsorbed following steps 2.1–2.8 until the antibody is completely absorbed.

[0092] 2.10 If the sample concentration is too high and precipitation occurs, centrifuge (3000 rpm for 10 minutes) to remove the precipitate. Measure the light absorption of the final eluent.

[0093] 2.11 Purified serum samples were retained for ELISA detection.

[0094] Example 7: Detection of the phosphorylation specificity of this antibody by ELSIA method 1. Coating: Add 20 μL of protein and 5 μL of peptide to 1 ml of coating buffer and mix thoroughly using a vortex mixer. Add 150 μL to a polyethylene plate and coat at 37°C for 2 hours. After coating, wash three times with PBST for five minutes each time.

[0095] 2. Sealing: Add 150 μL of sealing agent and seal at 37°C for one hour. After sealing, wash three times with Pbst for five minutes each time.

[0096] 3. Primary antibody: Add 4 μL of antibody to 1 ml of blocking agent and mix thoroughly. Add 150 μL to a polyethylene plate and incubate at 37°C for one hour. Wash three times with PBST for five minutes each time.

[0097] 4. Secondary Antibody: Add rabbit antibody to the blocking agent (1:8000), mix well, add 150ul to the polyethylene plate, and incubate at 37 degrees Celsius for one hour. Wash three times with Pbst, five minutes each time.

[0098] 5. Color development: Mix color developer A and color developer B (1:1), place in the dark, take 125ul, and place in the dark for 10 minutes.

[0099] 6. Termination: Add 2m of sulfuric acid. 7. Results: Measured using an ELISA reader. Table 3 Antibody dilution ratio 1:1000 Antibody dilution ratio 1:4000 Antibody dilution ratio 1:16000 Antibody dilution ratio 1:64000 N-Myristylated ARF1 Gly2 peptide 1.215 0.966 0.645 0.426 ARF1 peptide 0.212 0.109 0.095 0.008 Example 8 Immunohistochemistry Step 1. Baking slices The purpose of baking the slides is to firmly adhere the waxed tissue sections to the glass slide, preventing them from falling off during staining. This requires placing the sections in a constant temperature incubator at 56-60°C for at least 1 hour, typically 2-6 hours, until the wax on the tissue surface is completely melted, as observed by the naked eye. Because high-temperature drying can accelerate the oxidation of antigens in the tissue, baking at high temperatures can damage antigens, potentially leading to reduced positivity and incorrect staining localization.

[0100] Step 2. Dewaxing and hydration The tissue sections were immersed in xylene I and II for 15–30 minutes respectively to remove the wax. However, the xylene in the tissue is immiscible with the water-soluble staining solution, so it is necessary to further replace the xylene in the tissue using a descending gradient of ethanol. The sections were immersed in 100% ethanol I and II for 5 minutes each, and in 95%, 90%, 80%, and 70% ethanol for 2 minutes each. Rinse three times with PBS for 3 minutes each time, then place in distilled water for later use.

[0101] Step 3. Antigen Repair The recommended method is the high-temperature, high-pressure antigen retrieval method using citrate buffer, which is suitable for antigen retrieval in large quantities of neutral formalin-fixed and paraffin-embedded tissue sections. Its effectiveness is superior to microwave retrieval and direct boiling retrieval methods, resulting in more reliable immunohistochemical results. Method: Take a certain amount of pH 6.0 citrate buffer (800-1500 ml) in a pressure cooker and heat over high heat until boiling. Place the dewaxed and hydrated tissue sections on a stainless steel or heat-resistant plastic slide holder and immerse them in boiling buffer solution. Cover with a lid. Cover the pot, close the pressure valve, and continue heating until steam starts to escape. Start timing from when steam begins to escape. After 3-4 minutes, remove the pressure cooker from the heat source and let it cool. Once cooled to room temperature, remove the slide and rinse it twice with distilled water, then twice with PBS (pH=7.2-7.4), 3 times each time. Minutes (2 x 3').

[0102] Step 4. Perforating the cell membrane The slides were placed in 0.1%–0.3% Triton X-100 and soaked at room temperature for 25 minutes, followed by rinsing three times with PBS for 5 minutes each time. Triton X-100 is a detergent; its lipid solubility can perforate the cell membrane, increasing its permeability to antibodies. This step can be omitted when detecting cell membrane antigens.

[0103] Step 5. Inactivate endogenous enzymes and block endogenous biotin. The slides were immersed in 3% H2O2 for 20 minutes, then rinsed three times with PBS for 5 minutes each time. Peroxidases and catalases are present in some cells, most commonly in erythrocytes (hemoglobin), followed by rhabdomyosarcoma cells (myosin), granulocytes and monocytes (cytochrome), and hepatocytes and kidney cells (catalase). Endogenous H2O2 enzymes cause the substrate H2O2 to decompose, leading to DAB precipitation. Note: 3% H2O2 should be prepared fresh for each use, and the PBS used for washing with H2O2 should be separate from the PBS used for washing other materials. Using methanol to prepare hydrogen peroxide may be better than double-distilled water or PBS for protecting antigens and fixing tissues; however, prolonged incubation with hydrogen peroxide can easily cause slide detachment.

[0104] Step 6. Blocking with non-immune serum Antibodies can be adsorbed onto charged collagen and connective tissue components in tissue sections, leading to background staining. To prevent this, it is best to block the charge with a non-immune serum of the same species as the secondary antibody before treating the sections with the specific antibody, preventing the primary antibody from binding and inhibiting non-specific background staining. A common method is to use 2%–10% sheep serum or 2%–5% bovine serum albumin at room temperature for 10–30 minutes. However, it should be noted that this binding is not strong, so it is best not to rinse; simply discard the excess liquid and add the primary antibody directly. Add the specific primary antibody to the section and incubate overnight at 4°C (after removing from the refrigerator, it can be warmed to 37°C for 45 minutes), or incubate at 37°C for 1–2 hours. Afterward, rinse three times with PBS, 5 minutes each time. Specific antibodies are commonly diluted with PBS containing 1%–3% non-immune serum and should be prepared and used immediately. Diluted primary antibodies should not be stored for more than three days. The content of the target protein varies in different tissues; therefore, antibody dilution ratios should be determined by serial dilution according to the antibody's instructions to determine the optimal concentration. Note: Do not use dry slides. The reagent should fully cover the tissue, extending 2mm beyond the edge. When using a PAP pen, extend 3-4mm beyond the edge.

[0105] Step 8. Secondary Antibiotic Incubation Add appropriately diluted enzyme-labeled secondary antibody to the slide and incubate at room temperature for 30-60 minutes, followed by rinsing three times with PBS for 5 minutes each time. Secondary antibodies are available in anti-mouse, anti-rabbit, and anti-goat varieties, and their specificity must match the primary antibody. Otherwise, incorrect ligation between the primary and secondary antibodies can lead to false-negative staining results. For example, rabbit anti-mouse or human primary antibodies must be matched with anti-rabbit secondary antibodies, and mouse anti-rat or human primary antibodies must be matched with anti-mouse secondary antibodies. Note: If the secondary antibody is biotinylated, streptavidin-HRP should also be added.

[0106] Step 9. Color Development Color development is the final and crucial step in immunohistochemical staining. Generally, DAB or AEC is used for peroxidase detection systems. The former produces a brown color, while the latter produces a red color. To obtain optimal color development, strict microscopic control is essential, stopping when the detected substance reaches its strongest color and the background becomes colorless. Based on experience, DAB should not be left standing for more than 30 minutes after preparation; it cannot be used after this time. When DAB is added to tissue sections, the reaction time should not exceed 10 minutes (ideally within 5 minutes); otherwise, the reaction should be terminated regardless of whether a positive result is obtained. Background color is easily observed when using DAB to develop tissues rich in endogenous enzymes; therefore, microscopic control should be implemented as early as possible to achieve optimal resolution. DAB has carcinogenic effects; therefore, gloves should be worn during handling, and skin contact should be minimized. Hands should be washed promptly after use, and laboratory supplies that have come into contact with DAB should ideally be soaked in washing solution for 24 hours before reuse. The drawback of the AEC staining system is its high solubility in organic solvents; therefore, aqueous mounting media should be used primarily, and stained sections cannot be stored for long periods. During immunoenzyme staining, increasing the enzyme substrate concentration or prolonging the incubation period can enhance staining intensity. For peroxidase staining, a high H2O2 concentration can cause the reaction to proceed too quickly, leading to a darker background, and excessive H2O2 can inhibit enzyme activity; therefore, the H2O2 concentration should be moderate. If the above steps use a streptavidin-biotin-alkaline phosphatase complex, then NBT / BCIP should be used as the staining system. A positive result is blue-black. Rinse with distilled or tap water after staining. When using alkaline phosphatase as the label substrate, a 0.02 mol / L LTBS (pH 8.2) buffer is preferable during staining.

[0107] Step 10. Counterstain cell nuclei with hematoxylin. To ensure clear visualization of tissue structures and facilitate accurate localization, tissue sections are often counterstained. Hematoxylin is the most commonly used nuclear dye, but methyl green and nuclear fast red can also be used depending on the situation. Staining lasts approximately 10 seconds, with the degree of staining controlled under the microscope. If the results are satisfactory, rinsing with tap water will restore the blue color.

[0108] Step 11. Sealing If DAB staining is used, the sections are dehydrated by a gradient of ethanol (80% ethanol for 2 minutes, 95% ethanol for 2 minutes), ethanol I and 100% ethanol II for 5 minutes each, xylene I and II for 5 minutes each, and finally mounted with neutral resin. If AEC staining is used, the sections are not dehydrated by ethanol, and after rinsing and drying, they are mounted directly with water-based mounting medium.

[0109] The antibody was used for immunohistochemical analysis of paraffin sections of human oral squamous cell carcinoma at a dilution of 1:200. Results are as follows: Figure 1As shown, this antibody can be accurately used in IHC experiments to analyze the myristylation state of the N-terminal Gly2 of endogenous ARF1.

[0110] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A polypeptide for preparing ARF1 antibodies, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.2; or a polypeptide derived from the polypeptide shown in SEQ ID NO.2 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function.

2. A gene fragment, characterized in that: The gene fragment can encode the polypeptide as described in claim 1.

3. Use of the polypeptide of claim 1 for the preparation of ARF1 antibodies.

4. The use according to claim 3, characterized in that: The polypeptide was used to prepare ARF1 antibody after coupling.

5. A method for preparing ARF1 antibody, wherein the method involves immunizing an animal with the polypeptide to induce the production of ARF1 antibody, and then collecting the animal serum to obtain serum containing ARF1 antibody.

6. The method according to claim 5, characterized in that: The animal in question is either a rabbit or a sheep.

7. The method according to claim 5, characterized in that: The polypeptide is used to immunize animals after conjugation.

8. The method according to claim 5, characterized in that: The immunized animals include those receiving initial immunization and those receiving booster immunization.

9. The method according to claim 5, characterized in that: The method also includes extracting and purifying animal serum.

10. The method according to claim 9, characterized in that: The extraction and purification process employed a polypeptide cross-linking chromatography column to purify animal serum.

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