CN110982866A - Preparation and sealing method of protein powder for sealing Western blotting - Google Patents
Preparation and sealing method of protein powder for sealing Western blotting Download PDFInfo
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Abstract
The invention relates to a preparation method of fish gelatin protein powder for sealing a transfer film in western blotting, which is characterized in that PBS buffer solution is used for dissolving sea fish skin gelatin to prepare fish skin gelatin solution, and the fish skin gelatin solution is subjected to enzymolysis by using a mixed enzyme solution of bovine trypsin and thrombin freeze-dried powder, ultrafiltration, enrichment and drying to obtain the fish gelatin protein. The invention also discloses a protein immunoblotting blocking method. The invention effectively solves the common problems of poor sealing effect of the transfer membrane, high exposure background, detection of nonspecific strips and the like in a Western blotting experiment. By adopting the sealant for the Western blot experiment, after antibody incubation, specific protein detection bands can be obtained through ECL substrate detection, the background is clean and has no impurity band, and meanwhile, the incubation time is shortened to 15min, so that a technical basis is provided for obtaining accurate and efficient experimental results.
Description
Technical Field
The invention belongs to the technical field of protein research, and particularly relates to a preparation method of fish gelatin protein powder for sealing a transfer printing film in western blotting; the invention also discloses a protein immunoblotting sealing method.
Background
The Western Blot (Western Blot) technique is a method in which a protein sample is separated by polyacrylamide gel electrophoresis, transferred to a solid support (e.g., nitrocellulose membrane), and an antigen-antibody reaction is used to detect a target protein. It is a commonly used experimental method in molecular biology, biochemistry and immunogenetics.
The conventional Western Blot operation scheme comprises the following steps: preparing a protein sample, carrying out electrophoretic separation, transferring a membrane, sealing, washing, incubating with a primary antibody, washing, incubating with a secondary antibody, washing and detecting. However, the method involves many steps and factors, so to obtain a better experimental result, the following factors are often affected: accurate quantification of protein samples, preparation of protein gel, transfer printing conditions, electrophoresis condition setting, selection of buffer solution, selection of confining solution and confining conditions, specificity of antibody, chemiluminescence developing solution and the like.
Adequate sealing of the transfer membrane is a key factor in obtaining accurate protein assays. Currently, common blocking buffers used in laboratories are: 5% skimmed milk powder, 5% BSA and other protein components from mammalian bovine sources. The main source of the antibody used in the Western Blot experiment is also mammals, and even if a specific antibody is used, the risk of cross reaction of the antibody and a blocking solution is increased, so that the detection of a high background and a non-specific band after color development interferes with the accuracy of the experiment.
BSA, which is called Bovine Serum Albumin (BSA) is used as a blocking solution, is a globulin in bovine serum, comprises 607 amino acid residues, has a molecular weight of 66.446KDa, and has an isoelectric point of 4.7. BSA is a blocking solution with good blocking effect in the existing WB experiment, but the BSA is expensive and has high experiment cost. The price of BSA is about 100 times of the price of common skim milk; moreover, the BSA source is mammalian, and interference or pollution can be caused in a homologous WB experiment.
The skim milk powder is used as a sealing liquid, and is prepared by removing fat from fresh milk and drying, wherein the fat can be reduced to about 1% and other changes are small. The skimmed milk powder is milk white or light yellow, has mild Olibanum and sweet taste, and has no deterioration, smell or odor. A powdered product having not less than 34% protein and not more than 2.0% fat based on milk solids not containing fat. The skimmed milk powder has the advantage of low cost. The defect is that the protein content of the skimmed milk powder is not high, and compared with the protein content of BSA (bovine serum albumin) which is up to 90%, the protein content of the skimmed milk powder is only more than 30%; furthermore, the skim milk powder is still mainly from mammals, and in the homologous WB experiment, interference or pollution can be caused. Therefore, the research and development of new protein powder for sealing is of great significance.
Disclosure of Invention
The invention aims to provide a preparation method of fish gelatin protein powder for sealing a transfer printing film in a protein immunoblot, aiming at the defects of the prior art.
The invention also aims to provide a method for carrying out Western blot blocking on the fish gelatin protein powder prepared by the method.
The technical problem to be solved by the present invention is achieved by the following technical means. The invention relates to a preparation method of fish gelatin protein powder for sealing a transfer printing film in western blotting, which is characterized by comprising the following steps:
(1) dissolving sea fish skin gelatin by using PBS buffer solution to prepare a fish skin gelatin solution with the mass concentration of 10-50%;
(2) preparing 1mg/mL mixed enzyme solution from bovine trypsin and thrombin lyophilized powder according to the weight ratio of 1:10 to 10:1 by using sterilized purified water for later use;
(3) keeping the temperature of the prepared fish gelatin solution at 37 ℃, and stirring at 50-100 rpm;
(4) slowly adding a proper amount of prepared mixed enzyme solution, and carrying out heat preservation reaction at 37 ℃ for 6 hours;
(5) heating to 60 deg.C, maintaining the temperature for 30min to inactivate enzyme, and finishing enzymolysis reaction;
(6) treating the reaction solution by using a two-stage ultrafiltration membrane system, wherein the first-stage membrane enables small molecular proteins to permeate through and is used for enriching hydrolyzed gelatin and proper active enzyme for post-treatment; concentrating the protein by a secondary membrane until the concentration of the protein is 50%;
(7) spray drying to obtain fish gelatin protein.
The invention relates to a preparation method of fish gelatin protein powder for sealing a transfer printing film in western blotting, which further adopts the preferable technical scheme that: in the step (1): the mass concentration of the fish skin gelatin solution is 15-25%.
The invention relates to a preparation method of fish gelatin protein powder for sealing a transfer printing film in western blotting, which further adopts the preferable technical scheme that: in the step (2): preparing the bovine trypsin and thrombin freeze-dried powder into a mixed enzyme solution according to the weight ratio of 2-5: 1.
The invention also discloses a protein immunoblotting blocking method, which uses the fish gelatin protein powder prepared by the technical scheme as a blocking liquid raw material and comprises the following specific steps:
(1) extracting a protein sample: taking a freshly cultured A549 cell, removing a culture solution, and washing the cell by using PBS; adding 200 mul RIPA cell lysate containing PMSF with final concentration of 1mM into each well of a 6-well plate, blowing with a gun for several times to make the lysate and cells fully contact and lyse the cells; centrifuging the lysed cell sample at 14,000g for 5min, taking the supernatant, subpackaging the supernatant into a PCR tube, and standing at-80 ℃ for later use;
(2) protein quantification and pre-loading treatment: taking the protein extract prepared in the step (1) and determining the protein concentration by adopting a BCA method; adding a protein loading buffer solution with the mass volume ratio of 5 times to the sample with the determined protein concentration, fully and uniformly mixing, placing in a metal bath at 95 ℃ for denaturation for 10min, and placing at-80 ℃ for standby instantly;
(3) preparing gel: preparing 10% SDS-PAGE separation gel and 5% SDS-PAGE concentrated gel according to the molecular weight of the protein, and immediately filling gel after TEMED is added;
(4) glue pouring: fixing a clean glass plate, adding a prepared separation gel, and adding absolute ethyl alcohol to seal and remove bubbles; after the gel is solidified, absorbing the absolute ethyl alcohol in the glass plate by using filter paper, then adding the concentrated gel and inserting the gel into a comb, and slightly pulling out the comb after the gel is solidified again;
(5) electrophoresis: placing the SDS-polyacrylamide gel prepared in the step (4) into an electrophoresis tank, adding TGS electrophoresis buffer solution into the electrophoresis tank, sampling 10 mu L of the A549 cell lysate with the concentration of 2.4mg/mL prepared in the step (2), and starting electrophoresis; the voltage of the concentrated gel is 80V, the voltage of the separation gel is 130V, and electrophoresis is finished when the strip reaches the bottom of the glass plate;
(6) film transfer: applying the separation gel obtained in the step (5) after electrophoresis to a transfer membrane; activating the PVDF membrane in methanol for 3min, putting the black surface of the clamp into a glass plate filled with transfer liquid, sequentially putting a layer of sponge pad, three layers of filter paper, separation glue, the PVDF membrane and three layers of filter paper from bottom to top, finally covering a layer of sponge pad, removing bubbles, clamping the clamp, adding a precooled membrane transferring buffer solution, setting the current to be 200 mA, and transferring the membrane in an ice bath for 100 min;
(7) and (3) sealing: cutting the PVDF film which is converted in the step (6) into 4 pieces, sealing according to the following sealing liquid and sealing conditions, and sealing for 15min at room temperature by using 10% fish gelatin protein powder sealing liquid; 5% skim milk, sealing at room temperature for 1 h;
(8) primary antibody incubation: taking the membrane sealed in the step (7) for primary antibody incubation; diluting the primary antibody with a selected GAPDH specific antibody at a ratio of 1:10000 according to the recommendation of a specification, diluting the primary antibody with a diluent to a final concentration of 0.1 mug/mL, putting the sealed PVDF membrane into an incubation box, and incubating overnight at 4 ℃ to complete subsequent experiments; washing with TBST for 5 times, 5min each time;
(9) and (3) secondary antibody incubation: taking the membrane incubated with the primary antibody in the step (8) for secondary antibody incubation; selecting a goat anti-mouse IgG antibody marked by HRP (horse radish peroxidase) as a secondary antibody, diluting the secondary antibody by 1:5000 according to the instruction recommendation, and diluting the secondary antibody by using a corresponding blocking buffer solution; incubating the membrane in a secondary antibody for 1 h; washing with TBST for 5 times, 5min each time;
(10) and (3) chemiluminescence detection: adding the ultra-sensitive ECL luminescence base solution into the membrane incubated by the antibody in the step (9), putting the membrane into a gel imaging system after the membrane is fully contacted with the luminescence base solution, and setting different exposure times: 1s, 30s, 120s, and detecting.
The invention relates to a protein immunoblotting blocking method, which adopts a further preferable technical scheme that in the step (1), the final concentration of each component of the protein lysate is as follows: 150 mmoL/L NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmoL/L Tris (pH8.0), stored at room temperature and dissolved in a water bath at 60 ℃ before use.
The invention relates to a protein immunoblotting blocking method, which further preferably adopts the technical scheme that the PMSF solution is prepared as follows: PMSF was dissolved in isopropanol to a final concentration of 10mM, dissolved and stored in aliquots at-20 ℃.
The invention relates to a protein immunoblotting blocking method, which adopts a further preferable technical scheme that the protein loading buffer solution is prepared from 50% of glycerol, 250mM Tris & Cl (pH6.8), 10% of β -mercaptoethanol, 1% of bromophenol blue and 10% of SDS.
The invention relates to a protein immunoblotting blocking method, which further adopts the preferable technical scheme that 5% SDS-PAGE concentrated gel is prepared as follows: preparation of 9ml required 6ml H2O, 1.5ml of 30% acrylamide (29: 1), 1.5ml of 1M TRIS-HCl (pH6.8), 120. mu.l of 10% SDS, 90. mu.l of 10% AP, 12. mu.l of TEMED;
the 10% SDS-PAGE gels were prepared as follows: preparation of 15ml required 5.9ml H2O, 5ml of 30% acrylamide (29: 1), 3.8ml of 1.5M TRIS-HCl (pH 8.8), 150. mu.l of 10% SDS, 150. mu.l of 10% AP, 7.5. mu.l of TEMED.
The invention relates to a protein immunoblotting blocking method, which adopts a further preferable technical scheme that the TGS electrophoresis buffer solution is prepared as follows: 3g of Tris-base, 14.4g of glycine14, 0.1% SDS (10 ml of 10% SDS) is needed for preparing 1L of TGS protein electrophoresis buffer;
the transfer buffer is prepared as follows: 3g of Tris-base, 14.4g of glycine and 200ml of methanol are needed for preparing 1L of membrane conversion buffer solution;
the TBST was prepared as follows: 25mM Tris, 0.15mM NaCl, 0.05% Tween-20, pH7.5; the 5% skim milk is prepared as follows: preparing 100mL of 5% skim milk, namely 5g of skim milk, and dissolving the skim milk in TBST buffer solution;
the primary anti-dilution solution is prepared as follows: 100mL of primary antibody dilution was prepared by dissolving 1g of BSA, 1mL of 5% sodium azide in TBST buffer.
The invention relates to a protein immunoblotting blocking method, which further adopts the preferable technical scheme that the preparation method of the 10% fish gelatin protein liquid comprises the following steps: 10g of fish gelatin protein powder is needed for each 100mL of 10% fish gelatin protein blocking solution, and the fish gelatin protein powder is dissolved in TBST buffer solution. The fish gelatin protein solution may also be dissolved using protein solvents commonly used in the art.
Compared with the prior art, the method has the following beneficial effects:
(1) the collagen extracted from the fish skin by the method is mixed/hydrolyzed in stages to generate protein with different sizes, and compared with the traditional macromolecular skim milk mixed protein, the prepared protein has more sufficient sealing on a transfer printing film and clean exposure background.
(2) The fish gelatin protein prepared by the method has high sealing efficiency, and can be quickly sealed at room temperature for 15 minutes through experimental verification.
(3) The fish gelatin protein prepared by the method has strong specificity; the method is different from mammal-derived skim milk with the same source as the antibody, and can not generate non-specific reaction with the incubation antibody, so that the accuracy of the experimental result is enhanced.
(4) The fish gelatin protein prepared by the method has low cost, and compared with skimmed milk powder with higher price, the fish gelatin protein can be extracted from waste fish scales, and the preparation method is simple and has low cost.
(5) The fish gelatin protein prepared by the preparation method is used as a novel confining liquid applied to Western Blot experiments, and is subjected to mixing/segmented hydrolysis to generate protein with uneven molecular weight, so that the confining effect of the fish gelatin protein is superior to that of the traditional protein with fixed molecular weight. The extraction source of the fish gelatin protein is different from that of mammals, and the fish gelatin protein does not have cross reaction with antibodies, so that background reaction is avoided, and the specificity of experimental results is enhanced.
Drawings
FIG. 1 is a graph of the results of measurements of different exposure times after blocking a transfer membrane with 10% fish gelatin protein for 15min at room temperature and incubating with antibodies;
FIG. 2 is a graph showing the results of detection of different exposure times of antibody incubation after blocking the transfer film with 5% skim milk at room temperature for 1 h;
FIG. 3 is a graph of the detection results of commercial rapid blocking reagent A after blocking the transfer membrane for 15min at room temperature and incubating with antibodies for different exposure times;
FIG. 4 is a graph of the detection results of commercial rapid blocking reagent B at room temperature for 15min after blocking the transfer membrane and incubating the transfer membrane with antibody for different exposure times.
Detailed Description
The following further describes particular embodiments of the present invention to facilitate further understanding of the present invention by those skilled in the art, and does not constitute a limitation to the right thereof.
Example 1, a method for preparing fish gelatin protein powder for blocking a transfer membrane in western blotting, comprising the following steps:
(1) dissolving sea fish skin gelatin by using PBS buffer solution to prepare a fish skin gelatin solution with the mass concentration of 15%;
(2) preparing 1mg/mL mixed enzyme solution from bovine trypsin and thrombin lyophilized powder according to the weight ratio of 3:1 by using sterilized purified water for later use;
(3) keeping the temperature of the prepared fish gelatin solution at 37 ℃, and stirring at 80 rpm;
(4) slowly adding a proper amount of prepared mixed enzyme solution, and carrying out heat preservation reaction at 37 ℃ for 6 hours;
(5) heating to 60 deg.C, maintaining the temperature for 30min to inactivate enzyme, and finishing enzymolysis reaction;
(6) treating the reaction solution by using a two-stage ultrafiltration membrane system, wherein the first-stage membrane enables small molecular proteins to permeate through and is used for enriching hydrolyzed gelatin and proper active enzyme for post-treatment; concentrating the protein by a secondary membrane until the concentration of the protein is 50%;
(7) spray drying to obtain fish gelatin protein.
Embodiment 2, a method for preparing fish gelatin protein powder for blocking a transfer membrane in western blotting, comprising the following steps:
(1) dissolving sea fish skin gelatin by using PBS buffer solution to prepare a fish skin gelatin solution with the mass concentration of 25%;
(2) preparing 1mg/mL mixed enzyme solution from bovine trypsin and thrombin lyophilized powder according to the weight ratio of 2:1 by using sterilized purified water for later use;
(3) keeping the temperature of the prepared fish gelatin solution at 37 ℃, and stirring at 100 rpm;
(4) slowly adding a proper amount of prepared mixed enzyme solution, and carrying out heat preservation reaction at 37 ℃ for 6 hours;
(5) heating to 60 deg.C, maintaining the temperature for 30min to inactivate enzyme, and finishing enzymolysis reaction;
(6) treating the reaction solution by using a two-stage ultrafiltration membrane system, wherein the first-stage membrane enables small molecular proteins to permeate through and is used for enriching hydrolyzed gelatin and proper active enzyme for post-treatment; concentrating the protein by a secondary membrane until the concentration of the protein is 50%;
(7) spray drying to obtain fish gelatin protein.
Example 3, a method for blocking a protein immunoblot, which uses the fish gelatin protein powder prepared in example 1 or 2 as a blocking liquid raw material, comprising the following steps:
(1) extracting a protein sample: taking a freshly cultured A549 cell, removing a culture solution, and washing the cell by using PBS; adding 200 mul RIPA cell lysate containing PMSF with final concentration of 1mM into each well of a 6-well plate, blowing with a gun for several times to make the lysate and cells fully contact and lyse the cells; centrifuging the lysed cell sample at 14,000g for 5min, taking the supernatant, subpackaging the supernatant into a PCR tube, and standing at-80 ℃ for later use;
(2) protein quantification and pre-loading treatment: taking the protein extract prepared in the step (1) and determining the protein concentration by adopting a BCA method; adding a protein loading buffer solution with the mass volume ratio of 5 times to the sample with the determined protein concentration, fully and uniformly mixing, placing in a metal bath at 95 ℃ for denaturation for 10min, and placing at-80 ℃ for standby instantly;
(3) preparing gel: preparing 10% SDS-PAGE separation gel and 5% SDS-PAGE concentrated gel according to the molecular weight of the protein, and immediately filling gel after TEMED is added;
(4) glue pouring: fixing a clean glass plate, adding a prepared separation gel, and adding absolute ethyl alcohol to seal and remove bubbles; after the gel is solidified, absorbing the absolute ethyl alcohol in the glass plate by using filter paper, then adding the concentrated gel and inserting the gel into a comb, and slightly pulling out the comb after the gel is solidified again;
(5) electrophoresis: placing the SDS-polyacrylamide gel prepared in the step (4) into an electrophoresis tank, adding TGS electrophoresis buffer solution into the electrophoresis tank, sampling 10 mu L of the A549 cell lysate with the concentration of 2.4mg/mL prepared in the step (2), and starting electrophoresis; the voltage of the concentrated gel is 80V, the voltage of the separation gel is 130V, and electrophoresis is finished when the strip reaches the bottom of the glass plate;
(6) film transfer: applying the separation gel obtained in the step (5) after electrophoresis to a transfer membrane; activating the PVDF membrane in methanol for 3min, putting the black surface of the clamp into a glass plate filled with transfer liquid, sequentially putting a layer of sponge pad, three layers of filter paper, separation glue, the PVDF membrane and three layers of filter paper from bottom to top, finally covering a layer of sponge pad, removing bubbles, clamping the clamp, adding a precooled membrane transferring buffer solution, setting the current to be 200 mA, and transferring the membrane in an ice bath for 100 min;
(7) and (3) sealing: cutting the PVDF film which is converted in the step (6) into 4 pieces, sealing according to the following sealing liquid and sealing conditions, and sealing for 15min at room temperature by using 10% fish gelatin protein powder sealing liquid; 5% skim milk, sealing at room temperature for 1 h;
(8) primary antibody incubation: taking the membrane sealed in the step (7) for primary antibody incubation; diluting the primary antibody with a selected GAPDH specific antibody at a ratio of 1:10000 according to the recommendation of a specification, diluting the primary antibody with a diluent to a final concentration of 0.1 mug/mL, putting the sealed PVDF membrane into an incubation box, and incubating overnight at 4 ℃ to complete subsequent experiments; washing with TBST for 5 times, 5min each time;
(9) and (3) secondary antibody incubation: taking the membrane incubated with the primary antibody in the step (8) for secondary antibody incubation; selecting a goat anti-mouse IgG antibody marked by HRP (horse radish peroxidase) as a secondary antibody, diluting the secondary antibody by 1:5000 according to the instruction recommendation, and diluting the secondary antibody by using a corresponding blocking buffer solution; incubating the membrane in a secondary antibody for 1 h; washing with TBST for 5 times, 5min each time;
(10) and (3) chemiluminescence detection: adding the ultra-sensitive ECL luminescence base solution into the membrane incubated by the antibody in the step (9), putting the membrane into a gel imaging system after the membrane is fully contacted with the luminescence base solution, and setting different exposure times: 1s, 30s, 120s, and detecting.
The final concentration of each component of the protein lysate is as follows: 150 mmoL/L NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmoL/L Tris (pH8.0), stored at room temperature and dissolved in a water bath at 60 ℃ before use.
The PMSF solution is prepared as follows: PMSF was dissolved in isopropanol to a final concentration of 10mM, dissolved and stored in aliquots at-20 ℃.
The protein loading buffer solution is prepared from 50% of glycerol, 250mM Tris & Cl (pH6.8), 10% of β -mercaptoethanol, 1% of bromophenol blue and 10% of SDS.
The 5% SDS-PAGE gel concentrate was prepared as follows: preparation of 9ml required 6ml H2O, 1.5ml of 30% acrylamide (29: 1), 1.5ml of 1M TRIS-HCl (pH6.8), 120. mu.l of 10% SDS, 90. mu.l of 10% AP, 12. mu.l of TEMED;
the 10% SDS-PAGE gels were prepared as follows: preparation of 15ml required 5.9ml H2O, 5ml of 30% acrylamide (29: 1), 3.8ml of 1.5M TRIS-HCl (pH 8.8), 150. mu.l of 10% SDS, 150. mu.l of 10% AP, 7.5. mu.l of TEMED.
The TGS electrophoresis buffer was prepared as follows: 3g of Tris-base, 14.4g of glycine, 0.1% SDS (10 ml of 10% SDS) is required for preparing 1L of TGS protein electrophoresis buffer;
the transfer buffer is prepared as follows: 3g of Tris-base, 14.4g of glycine and 200ml of methanol are needed for preparing 1L of membrane conversion buffer solution;
the TBST was prepared as follows: 25mM Tris, 0.15mM NaCl, 0.05% Tween-20, pH7.5; the 5% skim milk is prepared as follows: preparing 100mL of 5% skim milk, namely 5g of skim milk, and dissolving the skim milk in TBST buffer solution;
the primary anti-dilution solution is prepared as follows: 100mL of primary antibody dilution was prepared by dissolving 1g of BSA, 1mL of 5% sodium azide in TBST buffer.
The preparation method of the 10% fish gelatin protein liquid comprises the following steps: 10g of fish gelatin protein powder is needed for each 100mL of 10% fish gelatin protein blocking solution, and the fish gelatin protein powder is dissolved in TBST buffer solution.
Example 4 western blot blocking method comparative experiment:
1. reagent preparation
(1) The final concentrations of the components of the protein lysate for preparing the protein lysate are as follows: 150 mmoL/L NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmoL/L Tris (pH8.0), stored at room temperature and dissolved in a water bath at 60 ℃ before use.
(2) Preparation of PMSF solution PMSF was dissolved in isopropanol to a final concentration of 10mM and stored in aliquots at-20 ℃.
(3) A protein loading buffer was prepared, 5 Xthe protein loading buffer was composed of 50% glycerol, 250mM Tris-Cl (pH6.8), 10% β -mercaptoethanol, 1% bromophenol blue, 10% SDS. (4) SDS-polyacrylamide gel was divided into 2 types of gel concentrates and gels, 5% SDS-PAGE gel concentrate preparation requiring 6ml H2O, 1.5ml 30% acrylamide (29: 1), 1.5ml1M TRIS-HCl (pH6.8), 120. mu.l 10% SDS, 90. mu.l 10% AP, 12. mu.l TEMED was prepared for 9ml, and 10% SDS-gel isolate preparation requiring 5.9ml H2O, 5ml 30% acrylamide (29: 1), 3.8ml 1.5M Tris-HCl (pH 8.8), 150. mu.l 10% SDS, 150. mu.l 10% AP, 7.5. mu.l TEMED was prepared for 15 ml.
(5) Preparation of TGS running buffer formulation 1L of TGS protein running buffer required 3g of Tris-base, 14.4g of glycine, 0.1% SDS (10 ml of 10% SDS).
(6) Preparation of Membrane transfer buffer solution for Wet transfer 1L of Membrane transfer buffer solution for Wet transfer required 3g of Tris-base, 14.4g of glycine and 200ml of methanol.
(7) Preparation of TBST wash buffer final concentrations of each component: 25mM Tris, 0.15mM NaCl, 0.05% Tween-20, pH7.5.
(8) Preparation of 5% skim milk 100mL of 5% skim milk was prepared by dissolving 5g of skim milk in TBST buffer.
(9) Preparation of 10% Fish gelatin 10g of fish gelatin to be hydrolyzed were prepared into 100mL of 10% fish gelatin blocking solution and dissolved in TBST buffer.
(10) Preparation of primary anti-dilution preparation 100mL of primary anti-dilution required 1g BSA, 1mL 5% sodium azide, dissolved in TBST buffer.
2. The comparison of the blocking effect of different blocking solutions in the Western Blot experiment comprises the following steps:
(1) extracting a protein sample: freshly cultured a549 cells were removed from the culture medium and the cells were washed with PBS. Mu.l of RIPA cell lysate (containing 1mM PMSF at the final concentration) was added to each well of the 6-well plate, and the lysate was brought into contact with the cells by pipetting and the cells were lysed. Centrifuging the lysed cell sample at 14,000g for 5min, taking the supernatant, subpackaging the supernatant into a PCR tube, and standing at-80 ℃ for later use;
(2) protein quantification and pre-loading treatment: and (3) taking the protein extract prepared in the step (1) and determining the protein concentration by using a BCA method. Adding a protein loading buffer solution with the mass volume ratio of 5 times to the sample with the determined protein concentration, fully and uniformly mixing, placing in a metal bath at 95 ℃ for denaturation for 10min, and placing at-80 ℃ for standby instantly;
(3) preparing gel: preparing 10% SDS-PAGE separation gel and 5% SDS-PAGE concentrated gel according to the molecular weight of the protein, and immediately filling gel after TEMED is added to prevent gelling;
(4) glue pouring: fixing the clean glass plate, adding the prepared separation gel, and adding absolute ethyl alcohol to seal and remove bubbles. After the gel is solidified, absorbing the absolute ethyl alcohol in the glass plate by using filter paper, then adding the concentrated gel and inserting the gel into a comb, and slightly pulling out the comb after the gel is solidified again;
(5) electrophoresis: and (5) placing the SDS-polyacrylamide gel prepared in the step (4) in an electrophoresis tank, adding TGS electrophoresis buffer solution into the electrophoresis tank, sampling 10 mu L of the A549 cell lysate with the concentration of 2.4mg/mL prepared in the step (2), and starting electrophoresis. The voltage of the concentrated gel is 80V, the voltage of the separation gel is 130V, and electrophoresis is finished when the strip reaches the bottom of the glass plate;
(6) film transfer: and (5) applying the separation gel subjected to electrophoresis in the step (5) to membrane transfer. Activating the PVDF membrane in methanol for 3min, putting the black surface of the clamp into a glass plate filled with transfer liquid, sequentially putting a layer of sponge pad, three layers of filter paper, separation glue, the PVDF membrane and three layers of filter paper from bottom to top, finally covering a layer of sponge pad, removing bubbles, clamping the clamp, adding a precooled membrane transferring buffer solution, setting the current to be 200 mA, and transferring the membrane in an ice bath for 100 min;
(7) and (3) sealing: cutting the PVDF film which is converted in the step (6) into 4 pieces, sealing according to the following sealing liquid and sealing conditions, respectively, sealing by 10% fish gelatin protein sealing liquid and a commercial rapid sealing reagent A, B for 15min at room temperature; 5% skim milk, sealing at room temperature for 1 h;
(8) primary antibody incubation: and (7) taking the membrane after the sealing for primary antibody incubation. And (3) selecting a GAPDH specific antibody of a Wuhan Sanying rat source according to the instruction recommendation, diluting the antibody by 1:10000 by using a primary antibody diluent, diluting the diluted antibody to a final concentration of 0.1 mug/mL, putting the sealed PVDF membrane into an incubation box, and incubating overnight at 4 ℃ to complete the subsequent experiment. Washing with TBST for 5 times, 5min each time;
(9) and (3) secondary antibody incubation: and (5) taking the membrane after the primary antibody is incubated in the step (8) for secondary antibody incubation. The secondary antibody is goat anti-mouse IgG antibody marked by Wuhan Sanying HRP, diluted at 1:5000 according to the instruction recommendation, and diluted by using a corresponding blocking buffer. The membrane was incubated in secondary antibody for 1 h. Washing with TBST for 5 times, 5min each time;
(10) and (3) chemiluminescence detection: adding the ultra-sensitive ECL luminescence base solution into the membrane incubated by the antibody in the step (9), putting the membrane into a gel imaging system after the membrane is fully contacted with the luminescence base solution, and setting different exposure times: 1s, 30s, 120s, and detecting.
The results are shown in FIGS. 1-4. The result shows that the method effectively solves the common problems of poor sealing effect of the transfer membrane, high exposure background, detection of nonspecific strips and the like in a Western blotting experiment. By adopting the sealant for the Western blot experiment, after antibody incubation, specific protein detection bands can be obtained through ECL substrate detection, the background is clean and has no impurity band, and meanwhile, the incubation time is shortened to 15min, so that a technical basis is provided for obtaining accurate and efficient experimental results.
Claims (10)
1. A preparation method of fish gelatin protein powder for sealing a transfer printing film in western blotting is characterized by comprising the following steps:
(1) dissolving sea fish skin gelatin by using PBS buffer solution to prepare a fish skin gelatin solution with the mass concentration of 10-50%;
(2) preparing 1mg/mL mixed enzyme solution from bovine trypsin and thrombin lyophilized powder according to the weight ratio of 1:10 to 10:1 by using sterilized purified water for later use;
(3) keeping the temperature of the prepared fish gelatin solution at 37 ℃, and stirring at 50-100 rpm;
(4) slowly adding a proper amount of prepared mixed enzyme solution, and carrying out heat preservation reaction at 37 ℃ for 6 hours;
(5) heating to 60 deg.C, maintaining the temperature for 30min to inactivate enzyme, and finishing enzymolysis reaction;
(6) treating the reaction solution by using a two-stage ultrafiltration membrane system, wherein the first-stage membrane enables small molecular proteins to permeate through and is used for enriching hydrolyzed gelatin and proper active enzyme for post-treatment; concentrating the protein by a secondary membrane until the concentration of the protein is 50%;
(7) spray drying to obtain fish gelatin protein.
2. The method for preparing the fish gelatin protein powder for blocking the transfer membrane in the western blotting according to claim 1, wherein in the step (1): the mass concentration of the fish skin gelatin solution is 15-25%.
3. The method for preparing the fish gelatin protein powder for blocking the transfer membrane in the western blotting according to claim 1, wherein in the step (2): preparing the bovine trypsin and thrombin freeze-dried powder into a mixed enzyme solution according to the weight ratio of 2-5: 1.
4. A protein immunoblotting blocking method, which is characterized in that the method uses the fish gelatin protein powder prepared in the claim 1 or 2 or 3 as a raw material of a blocking liquid, and comprises the following specific steps:
(1) extracting a protein sample: taking a freshly cultured A549 cell, removing a culture solution, and washing the cell by using PBS; adding 200 mul RIPA cell lysate containing PMSF with final concentration of 1mM into each well of a 6-well plate, blowing with a gun for several times to make the lysate and cells fully contact and lyse the cells; centrifuging the lysed cell sample at 14,000g for 5min, taking the supernatant, subpackaging the supernatant into a PCR tube, and standing at-80 ℃ for later use;
(2) protein quantification and pre-loading treatment: taking the protein extract prepared in the step (1) and determining the protein concentration by adopting a BCA method; adding a protein loading buffer solution with the mass volume ratio of 5 times to the sample with the determined protein concentration, fully and uniformly mixing, placing in a metal bath at 95 ℃ for denaturation for 10min, and placing at-80 ℃ for standby instantly;
(3) preparing gel: preparing 10% SDS-PAGE separation gel and 5% SDS-PAGE concentrated gel according to the molecular weight of the protein, and immediately filling gel after TEMED is added;
(4) glue pouring: fixing a clean glass plate, adding a prepared separation gel, and adding absolute ethyl alcohol to seal and remove bubbles; after the gel is solidified, absorbing the absolute ethyl alcohol in the glass plate by using filter paper, then adding the concentrated gel and inserting the gel into a comb, and slightly pulling out the comb after the gel is solidified again;
(5) electrophoresis: placing the SDS-polyacrylamide gel prepared in the step (4) into an electrophoresis tank, adding TGS electrophoresis buffer solution into the electrophoresis tank, sampling 10 mu L of the A549 cell lysate with the concentration of 2.4mg/mL prepared in the step (2), and starting electrophoresis; the voltage of the concentrated gel is 80V, the voltage of the separation gel is 130V, and electrophoresis is finished when the strip reaches the bottom of the glass plate;
(6) film transfer: applying the separation gel obtained in the step (5) after electrophoresis to a transfer membrane; activating the PVDF membrane in methanol for 3min, putting the black surface of the clamp into a glass plate filled with transfer liquid, sequentially putting a layer of sponge pad, three layers of filter paper, separation glue, the PVDF membrane and three layers of filter paper from bottom to top, finally covering a layer of sponge pad, removing bubbles, clamping the clamp, adding a precooled membrane transferring buffer solution, setting the current to be 200 mA, and transferring the membrane in an ice bath for 100 min;
(7) and (3) sealing: cutting the PVDF film which is converted in the step (6) into 4 pieces, sealing according to the following sealing liquid and sealing conditions, and sealing for 15min at room temperature by using 10% fish gelatin protein powder sealing liquid; 5% skim milk, sealing at room temperature for 1 h;
(8) primary antibody incubation: taking the membrane sealed in the step (7) for primary antibody incubation; diluting the primary antibody with a selected GAPDH specific antibody at a ratio of 1:10000 according to the recommendation of a specification, diluting the primary antibody with a diluent to a final concentration of 0.1 mug/mL, putting the sealed PVDF membrane into an incubation box, and incubating overnight at 4 ℃ to complete subsequent experiments; washing with TBST for 5 times, 5min each time;
(9) and (3) secondary antibody incubation: taking the membrane incubated with the primary antibody in the step (8) for secondary antibody incubation; selecting a goat anti-mouse IgG antibody marked by HRP (horse radish peroxidase) as a secondary antibody, diluting the secondary antibody by 1:5000 according to the instruction recommendation, and diluting the secondary antibody by using a corresponding blocking buffer solution; incubating the membrane in a secondary antibody for 1 h; washing with TBST for 5 times, 5min each time;
(10) and (3) chemiluminescence detection: adding the ultra-sensitive ECL luminescence base solution into the membrane incubated by the antibody in the step (9), putting the membrane into a gel imaging system after the membrane is fully contacted with the luminescence base solution, and setting different exposure times: 1s, 30s, 120s, and detecting.
5. The method for blocking western blot of protein according to claim 4, wherein in step (1), the final concentrations of the components of the protein lysate are as follows: 150 mmoL/L NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmoL/L Tris (pH8.0), stored at room temperature and dissolved in a water bath at 60 ℃ before use.
6. The method for blocking western blot of protein according to claim 4, wherein the PMSF solution is prepared as follows: PMSF was dissolved in isopropanol to a final concentration of 10mM, dissolved and stored in aliquots at-20 ℃.
7. The method of claim 4, wherein the protein loading buffer is prepared from 50% glycerol, 250mM Tris-Cl (pH6.8), 10% β -mercaptoethanol, 1% bromophenol blue, and 10% SDS.
8. The Western blot blocking method according to claim 4, wherein the 5% SDS-PAGE gel concentrate is prepared as follows: preparation of 9ml required 6ml H2O, 1.5ml of 30% acrylamide (29: 1), 1.5ml of 1M TRIS-HCl (pH6.8), 120. mu.l of 10% SDS, 90. mu.l of 10% AP, 12. mu.l of TEMED;
the 10% SDS-PAGE gels were prepared as follows: preparation of 15ml required 5.9ml H2O, 5ml of 30% acrylamide (29: 1), 3.8ml of 1.5M TRIS-HCl (pH 8.8), 150. mu.l of 10% SDS, 150. mu.l of 10% AP, 7.5. mu.l of TEMED.
9. The western blot blocking method according to claim 4, characterized in that: the TGS electrophoresis buffer was prepared as follows: 3g of Tris-base, 14.4g of glycine, 0.1% SDS (10 ml of 10% SDS) is required for preparing 1L of TGS protein electrophoresis buffer;
the transfer buffer is prepared as follows: 3g of Tris-base, 14.4g of glycinee and 200ml of methanol are needed for preparing 1L of membrane transferring buffer solution;
the TBST was prepared as follows: 25mM Tris, 0.15mM NaCl, 0.05% Tween-20, pH7.5; the 5% skim milk is prepared as follows: preparing 100mL of 5% skim milk, namely 5g of skim milk, and dissolving the skim milk in TBST buffer solution;
the primary anti-dilution solution is prepared as follows: 100mL of primary antibody dilution was prepared by dissolving 1g of BSA, 1mL of 5% sodium azide in TBST buffer.
10. The western blot blocking method according to claim 4, wherein the 10% fish gelatin protein solution is prepared as follows: 10g of fish gelatin protein powder is needed for each 100mL of 10% fish gelatin protein blocking solution, and the fish gelatin protein powder is dissolved in TBST buffer solution.
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