CN111157603A - Quick-setting leakage-proof method for small amount of separation gel in plate making by polyacrylamide gel - Google Patents
Quick-setting leakage-proof method for small amount of separation gel in plate making by polyacrylamide gel Download PDFInfo
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- CN111157603A CN111157603A CN201911342906.0A CN201911342906A CN111157603A CN 111157603 A CN111157603 A CN 111157603A CN 201911342906 A CN201911342906 A CN 201911342906A CN 111157603 A CN111157603 A CN 111157603A
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Abstract
The invention discloses a rapid-setting leakage-proof method for a small amount of separation gel in a polyacrylamide gel plate manufacturing process. Uniformly mixing the separation glue and Tetramethylethylenediamine (TEMED) to obtain a leakage-blocking glue; and conventionally placing the new or old long and short glass plate on a vertical frame, adding the plugging glue along one corner of the upper edge of the long and short glass plate, lifting the vertical frame at the sample adding end, enabling the liquid to quickly reach the bottom corner of the opposite side of the long and short glass plate, flatly placing the vertical frame, and solidifying the liquid to form a plugging glue layer. And pouring out part of liquid separated out from the upper layer of the leaking stoppage glue layer and then sucking the liquid by using a filter paper strip. According to a conventional method, sequentially adding separation glue, concentration glue and an inserting comb to finish gel plate making. The invention has low requirements on long and short glass plates and simple preparation process. The method can be applied to the mass production of commercial polyacrylamide gel precast slabs and the development of new experimental reagents.
Description
Technical Field
The invention belongs to the technical field of preparation of plates made of polyacrylamide gel, and relates to a small-amount separation gel quick-setting leakage-proof method for plates made of polyacrylamide gel.
Background
Polyacrylamide gel electrophoresis is one of the most basic techniques for biochemical analysis, has played an important role in biomedicine since the report of the technique, and western blot, nucleic acid and proteome analysis based on the technique are particularly important for the research of life science. The technology is based on the preparation of a polyacrylamide gel plate, and the quality of the gel plate directly influences the later experimental operation and result analysis. Although the gel plate has a large number of commercial products, the specific manufacture of the polyacrylamide gel plate still needs to be continued in specific experimental requirements, preliminary contact experiments and teaching experiments in colleges and universities. One of the problems frequently encountered in the glue making process is glue leakage, which cannot be coagulated into a complete glue board, resulting in failure of the whole experiment.
Hu Cheng Xiang, Zhang Yu Chun, Li Lei, et al, in SDS-polyacrylamide gel electrophoresis technology improvement and its application, proposed to use 2cm wide transparent adhesive tape tightly sealed for 3/4 weeks (except for the sample loading side), without wrinkles and bubbles, then absorb l.5mL edge sealing adhesive (30% Acr-Bis 0.5mL,3mol/L Tris-HCl 3-3.5mL, 10% AP0.2mL, TEMED15 μ L) to seal once, the excess adhesive is discarded immediately. But is relatively complicated and the plugging effect is not ideal.
The method of sealing edges by using high-concentration agar is proposed by Sungxian, Xiagong, Shenjia, and people in the field of polyacrylamide gel preparation and electrophoresis and overcoming the problems, but the situation that a part of glue is not tightly adhered to an electrophoresis tank or a glass plate during sealing is still caused by the fact that the glass plate and the electrophoresis tank are not dry or the electrophoresis tank is fixed too tightly, a comb is difficult to smoothly insert after gel is poured, and the strength is slightly large, so that glue solution leaks.
The applicant of the invention explores a set of simple and practical method for plugging the separation gel in advance by rapid setting on the basis of years of experiments and according to relevant theories and practices, and solves the problem of gel leakage by a simple method through gel preparation comparison and electrophoresis result verification.
Disclosure of Invention
The invention provides a method for quickly condensing and preventing leakage of a small amount of separation gel in a polyacrylamide gel plate manufacturing process aiming at solving the technical problem.
The method comprises the following steps:
1) uniformly mixing the separation glue and Tetramethylethylenediamine (TEMED) to obtain a leakage-blocking glue;
the volume ratio of the separation gel to the TEMED is 250: (1-5);
for the existing mini gel plate, the separation gel is preferably 500. mu.L.
TEMED has less high temperature consumption and more low temperature consumption according to the indoor temperature. The amount of TEMED and room temperature can influence the setting time of leaking stoppage glue, and TEMED volume is many and high temperature can lead to solidifying too fast, and solidify too slowly and then can influence the experiment progress, handles according to particular case.
2) And conventionally placing the new or old long and short glass plate on a vertical frame, adding the plugging glue along one corner of the upper edge of the long and short glass plate, lifting the vertical frame at the sample adding end, enabling the liquid to quickly reach the bottom corner of the opposite side of the long and short glass plate, flatly placing the vertical frame, and solidifying the liquid to form a plugging glue layer. The leaking stoppage glue layer is fully paved between the two long and short glass plates.
3) And pouring out part of liquid separated out from the upper layer of the leaking stoppage glue layer and then sucking the liquid by using a filter paper strip.
4) According to a conventional method, sequentially adding separation glue, concentration glue and an inserting comb to finish gel plate making.
The mass concentration of acrylamide in the separation gel is 8-15%.
The invention has the beneficial effects that:
1. the requirements on the long and short glass plates are not high: the rubber plate can be manufactured by using long and short glass plates with long edges seriously worn.
2. The preparation process is simple.
3. The method can be applied to the mass production of commercial polyacrylamide gel precast slabs and the development of new experimental reagents.
Drawings
FIG. 1 is a comparison of the leakage of polyacrylamide gel from three sets of processes, DIY (Do It Youself, Long and short glass plates and gasket strips as OLD), NEW (New Long and short glass plates and gasket strips), OLD (Long and short glass plates and gasket strips after 1 year use);
FIG. 2 is a DIY polyacrylamide gel electrophoresis diagram, wherein the left side 1 is concentrated gel (5%), 2 is separation gel (10%), 3 is molecular Marker, and 4 is bromophenol blue; 5. leakage-blocking glue (10%); on the right side is the Biyuntian biotechnologies Limited color prestained protein molecular weight standard (P0068).
FIG. 3 is a western blot analysis of protein electrophoresis and actin protein expression; a is Coomassie brilliant blue staining, B is near-infrared fluorescence labeling antibody reaction, wherein channels 1, 2 and 3 are Jurkat cell extracted protein, and channels 4, 5 and 6 are A549 cell extracted protein; c is B fluorescence scanning grey value statistical analysis.
Detailed Description
The present invention is further analyzed with reference to the following specific examples.
Calf serum used in the following examples was purchased from Hangzhou ilex bioengineering materials, Inc.; the RPMI1640 culture medium containing 100U/mL of penicillin and 100 mu g/mL of streptomycin is a product of Hangzhou keno biological Co., Ltd; the sterile plastic culture bottle is a Shanghai BD Falcon product; protein Marker, pancreatin digestive juice, Western and IP cell lysate and protein loading buffer are purchased from Biyuntian biotechnology limited company; the BCA Protein Assay Kit is a Pierce product; SDS-PAGE gel preparation kit purchased from Google biology of Wuhan; PVDF membrane is Millipore product; the long and short glass plates and the filler strip of the glue preparation equipment are bio-rad products; rabbit anti-human actin antibodies were purchased from Hangzhou Huaan Biotechnology Ltd; DyLight800 labeled anti-rabbit fluorescent labeled secondary antibody was purchased from Beijing Western Meijie technologies, Inc.
Example 1:
1) uniformly mixing 500 mu L of separation glue and 2 mu L of Tetramethylethylenediamine (TEMED) to obtain the leakage-blocking glue;
2) and (3) conventionally placing the long and short glass plates with the edges of the long and short glass plates which are longer than 1 year in time and aged by the filler strips on a stand, quickly adding the plugging glue prepared in the step (1) into the plugging glue along one corner of the upper edge of the long and short glass plates, lifting the stand at the sample adding end, enabling the liquid to quickly reach the bottom corners of the opposite sides of the long and short glass plates, flatly placing the stand, and solidifying the liquid to form a plugging glue layer.
3) And pouring out part of liquid separated out from the upper layer of the leaking stoppage glue layer and then sucking the liquid by using a filter paper strip.
4) According to a conventional method, adding separation gel (acrylamide concentration is 10%), concentration gel (acrylamide concentration is 5%) and inserting a comb in sequence to finish gel plate making.
Comparative example 1:
using a long and short glass plate and a cushion gum of the same type as those of the glass plate of example 1, a separation gum (acrylamide concentration: 10%), a concentration gum (acrylamide concentration: 5%) and an insertion comb were added in this order according to a conventional method to complete gel plate formation.
Comparative example 2:
using a long and short glass plate manufactured by the same type as in example 1 and a cushion rubber for more than 1 year, a separating rubber (acrylamide concentration: 10%), a thickening rubber (acrylamide concentration: 5%) and an inserting comb were added in the same amounts as in example 1 in this order according to a conventional method to complete gel plate manufacturing.
Application example 1:
1) cell culture cell lines Jurkat and A549 were cultured in a cell culture medium of 10% calf serum-containing RPMI1640(100U/mL penicillin, 100U/mL streptomycin), incubated at 37 ℃ in an incubator with saturated humidity and 5% carbon dioxide, and were ready for use when the growth was in log phase.
2) The electrophoresis sample is prepared by trypsinizing the cell A549, directly sucking Jurkat, collecting in a test tube, centrifuging at 1000rpm for 5min, washing with PBS buffer solution once, cracking the cell according to the Western and IP cell lysate instructions, centrifuging to extract supernatant, mixing with protein loading buffer solution, and treating with boiling water for 5min for later use.
3) The plates prepared by using the polyacrylamide gels of example 1, comparative example 1 and comparative example 2 were designated as DIY group, NEW group and OLD group, respectively.
4) And (4) assembling an electrophoresis apparatus according to the conventional method, pulling out a comb and loading the sample for electrophoresis, and stopping electrophoresis when bromophenol blue runs to the leakage stoppage adhesive layer. And (3) stripping and shooting, cutting down actin strip glue and transferring the PVDF film, dyeing the rest glue strips with Coomassie brilliant blue, and shooting after decolorization. PVDF membrane in 5% skimmed milk TBST closed for 1h, TBST rinse 5min, repeat three times. Then the PVDF membrane is soaked in a rabbit anti-human actin (1: 1000) chromatographic cabinet at 4 ℃ overnight in a shaking table, and the PVDF membrane is taken out the next day and rinsed for 5min in TBST and repeated for three times. Transferring the PVDF membrane into a DyLight 800-labeled anti-rabbit fluorescent labeled secondary antibody (1: 15000), shaking the membrane in a dark place for 2h in a chromatographic cabinet at 4 ℃, rinsing the membrane in a dark place for 5min in TBST, and repeating the steps for three times. And finally, detecting and analyzing by an Odyssey near-infrared fluorescence scanner to determine the gray value of the target band.
5) Data processing cell lines Jurkat and A549 extract protein were analyzed in triplicate, actin grayscale values were statistically processed in excel tables, and percent data were reported using Chi's (chi-square)2) Checking, the grey value is checked by t, and
and (6) drawing a diagram.
Results of application example 1
1) The glue leakage evaluation standard of the glue leakage condition of the glue making plate is that polyacrylamide gel appears on the outer side between the glass plate and the filler strip, and the separation line of the separation glue and the concentrated glue moves downwards and is uneven, so that the quality of the glue making plate is poor.
The three groups of DIY, NEW and OLD glue making equipment are all from the same product, and after the DIY is used for 1 year as the OLD group, the edges of the long and short glass platesGaps appear and the filler strip is aged. NEW is an unused long and short glass plate. Through the simultaneous operation, the rubber plates are configured for 30 times once a day and continuously for one month, the OLD composition power (60%) is low, the two groups of DIY (93.3%) and NEW (96.7%) have less rubber leakage, and the rubber preparation quality is obviously better than that of the OLD group. Jingka square (X)2) The OLD group was statistically significantly different from the other two groups (P)<0.01) and DIY and NEW have no statistical significance (P)>0.05) (fig. 1).
2) After gel electrophoresis, directly observing the upper surface of the DIY whole rubber plate, a plurality of comb teeth are arranged, a sample adding hole is formed between every two comb teeth, two horizontal parallel folded rays can be seen downwards and are respectively positioned between concentrated glue and separation glue and between the separation glue and a small amount of leakage-proof separation glue, the rubber plate is divided into an upper region, a middle region and a lower region, the middle region is the separation glue, the separation glue accounts for two thirds of a bio-rad mini electrophoresis glass short plate, and the separation glue also accounts for the positions of bromophenol blue dye and a pre-dyed protein molecular weight standard Marker (figure 2). The bromophenol blue dye is in a horizontal bead shape, each bead image represents the image of a sample in a concentrated gel hole, the consistency is kept, the prestained protein molecular weight standard Marker is erected in a separation gel, 9 strips are doped with red and blue, the mixture is clearly visible, the shape of each strip is similar to that of a sample hole, the distribution and the color are consistent with the image provided by the kit, and only 10KD is not seen. In addition, the distance between 180KD and 130KD is relatively shortened, and the distance between 26KD and 17KD is relatively lengthened.
3) Staining and blot analysis DIY gel was transected from it, and the gel in which protein Marker43KD was present analyzed for actin expression, and another gel stained with Coomassie brilliant blue, and 6 protein bands of Jurkat and A549 were clearly and smoothly visualized without abnormal problems (FIG. 3A). The actin-expressed protein bands are in the same horizontal line and have similar size and shape (FIG. 3B). The actin content in Jurkat and a549 cells was close to each other with no statistical difference by statistical treatment of the grey value of each band (P ═ 0.069, fig. 3C).
4) The prices of the long glass plate, the short glass plate and the filler strip which are commonly used at present are consulted on the internet of the prices of related glue-making articles, and are checked by related dealers, the prices are relatively close to the following table (table 1), foreign brands are obviously higher than domestic products, and the prices are over several times. The DIY method created by the research does not need the tools, and the cost can be ignored.
TABLE 1 price list for small gel vertical electrophoresis rubber mat strip and glass plate
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (3)
1. The rapid-setting leakage-proof method for the small amount of separation gel in the process of manufacturing the plate by the polyacrylamide gel is characterized by comprising the following steps of:
1) uniformly mixing the separation glue and Tetramethylethylenediamine (TEMED) to obtain a leakage-blocking glue;
2) conventionally placing a new or old long and short glass plate on a vertical frame, adding a plugging glue along one corner of the upper edge of the long and short glass plate, lifting the vertical frame at the sample adding end to enable liquid to reach the bottom corners of the long and short glass plates opposite to the side, flatly placing the vertical frame, and solidifying the liquid to form a plugging glue layer;
3) then pouring out part of liquid precipitated on the upper layer of the leaking stoppage glue layer and drying the liquid by using a filter paper strip;
4) according to a conventional method, sequentially adding separation glue, concentration glue and an inserting comb to finish gel plate making.
2. The method for preventing leakage of polyacrylamide gel plate by quick setting of small amount of separation gel as claimed in claim 1, wherein the separation gel content in the leakage-stopping gel is 500 ul.
3. The method for preventing leakage of small amount of separation gel from polyacrylamide gel sheet as claimed in claim 1, wherein the volume ratio of separation gel to TEMED in step 1) is 250: (1-5).
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| CN201911342906.0A CN111157603A (en) | 2019-12-23 | 2019-12-23 | Quick-setting leakage-proof method for small amount of separation gel in plate making by polyacrylamide gel |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN2095421U (en) * | 1991-05-24 | 1992-02-05 | 中国人民解放军海军医学研究所 | Electrophoresis apparatus |
| US20020128415A1 (en) * | 2001-03-08 | 2002-09-12 | The Regents Of The University Of California | Polyacrylamide medium for the electrophoretic separation of biomolecules |
| CN101614695A (en) * | 2009-07-23 | 2009-12-30 | 杭州吉来生物技术有限公司 | A kind of integral electrophoretic apparatus that is used for gel electrophoresis |
| CN103149262A (en) * | 2013-01-11 | 2013-06-12 | 安徽中医学院第一附属医院 | Xinfeng capsule water-soluble protein molecular weight detecting method |
| CN103275174A (en) * | 2013-06-20 | 2013-09-04 | 国家林业局泡桐研究开发中心 | General type kit for fast separating and dyeing polyacrylamide gel protein |
| CN105232187A (en) * | 2011-09-09 | 2016-01-13 | 安多拉米诺科学公司 | Means for controlled sealing of endovascular devices |
| CN106568825A (en) * | 2016-11-11 | 2017-04-19 | 浙江农林大学 | Polyacrylamide gel preparation method capable of preventing solution leakage |
| CN106957397A (en) * | 2017-02-27 | 2017-07-18 | 杭州启明医疗器械有限公司 | Anti- all leak gel rubber materials and preparation method thereof |
-
2019
- 2019-12-23 CN CN201911342906.0A patent/CN111157603A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2095421U (en) * | 1991-05-24 | 1992-02-05 | 中国人民解放军海军医学研究所 | Electrophoresis apparatus |
| US20020128415A1 (en) * | 2001-03-08 | 2002-09-12 | The Regents Of The University Of California | Polyacrylamide medium for the electrophoretic separation of biomolecules |
| CN101614695A (en) * | 2009-07-23 | 2009-12-30 | 杭州吉来生物技术有限公司 | A kind of integral electrophoretic apparatus that is used for gel electrophoresis |
| CN105232187A (en) * | 2011-09-09 | 2016-01-13 | 安多拉米诺科学公司 | Means for controlled sealing of endovascular devices |
| CN103149262A (en) * | 2013-01-11 | 2013-06-12 | 安徽中医学院第一附属医院 | Xinfeng capsule water-soluble protein molecular weight detecting method |
| CN103275174A (en) * | 2013-06-20 | 2013-09-04 | 国家林业局泡桐研究开发中心 | General type kit for fast separating and dyeing polyacrylamide gel protein |
| CN106568825A (en) * | 2016-11-11 | 2017-04-19 | 浙江农林大学 | Polyacrylamide gel preparation method capable of preventing solution leakage |
| CN106957397A (en) * | 2017-02-27 | 2017-07-18 | 杭州启明医疗器械有限公司 | Anti- all leak gel rubber materials and preparation method thereof |
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