![SMOBIO/[QP5220] Q-PAGE™ TGN Precast Gel (Midi, 15 wells, 10%), 10 gels/Midi, 15 wells, 10%), 10 gels</span>
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Description
Q-PAGE™ TGN (Tris-Glycine Novel) Precast Gels are ready-to-use acrylamide gels for SDS-PAGE running in Tris-Glycine buffer system. With unique formula, Q-PAGE™ TGN Precast Gels perform enhanced speed, better separation, and longer shelf life as compared with conventional Laemmli Tris-HCl gels. The protein migration patterns in Q-PAGE™ TGN series, however, are similar with typical Laemmli Tris-HCl gels, and thus Q-PAGE™ TGN Precast Gels are compatible to traditional SDS-PAGE and subsequent analyses.
Q-PAGE™ TGN Precast Gels are available in gradient (4 to 15%) and fixed (10%) concentrations of polyacrylamide in 12- and 15-well formats. Two available cassette sizes, Mini (10 x 8.3 cm) and Midi (10 x 10 cm), are compatible with most popular protein electrophoresis systems. Q-PAGE™ Mini (QP4XXX) Gels are suitable for Bio-Rad® and other systems. Q-PAGE™ Midi (QP5XXX) Gels are suitable for Invitrogen® XCell SureLock® Mini-Cell, Invitrogen® Mini Gel Tank, Hoefer SE260, and other systems.
Key Features
User-friendly gel cassette:
Numbered and framed wells for sample loading
Labeled warning sign and green tape as reminder
Enhanced gel performance:
Enhanced gel electrophoresis speed
Better band separation
Stable for shipping at ambient temperature
Easy compatibility:
Available as homogeneous and adjusted gradient gels for a wide range of protein separation.
Compatible with most popular protein electrophoresis systems
Storage and stability
Store Q-PAGE™ Precast Gels at 4°C for periods up to 12 months.
Do not freeze Q-PAGE™ Precast Gels. Remove tape and comb before electrophoresis.
Technical
Quick running, clear bands
Q-PAGE™ TGN Precast Gel can separate protein in 19 minutes using 300 V.
QP5220 Specifications
Gel | TGN(Tris-Glycine-Novel) | |
Buffersystems | Tris-Glycine (Laemmli) | |
Features | Quickrunning, clear bands | |
Cassettesize | Midi Gel (10 X 10 cm) | |
Geldimensions | 8.1 x 8.1 x0.1 cm (W x L xthickness) cm | |
Electrophoresissystem | Mini Gel Tank XCell SureLock, Hoefer SE260 | |
Well format& Capacity | 15 wells, 28 μl/well | |
Gelpercentage | 10 % | |
Accessorytray | Productiondescription Tip card Gel remover Cassetteopener | |
Manual
Manual_Q-PAGE™ TGN Precast Gel, Midi
SDS
SDS_Q-PAGE™ Precast Gel
Migration pattern
Setting Up and Running Q-PAGE™ Midi Precast Gel
Removing Q-PAGE Midi Gel from cassette
Setting up gel/membrane sandwich for Western transfer
Recommendations/Tips for Gel Running
1. Remove comb and tape before adaption. 2. Use fresh 1X running buffer for the inner cathode chamber. 3. Rinse the wells before sample loading. 4. Try 200 V first, and optimize the voltage and running time if needed. Do not set voltage lower than 100 V.
Sample Preparation for SDS-PAGE
1. Mix protein sample with 2X sample buffer.
2. Heat the diluted samples at 95°C for 5 min or at 70°C for 10 min.
3. Cool the diluted samples to 4°C and spin down the water condensed on tube surface. (If there is high viscosity part at bottom of tube, transfer supernatant to a new tube.)
Prepare Q-PAGE™ for Sample Loading
1.Open the blister tray of Q-PAGE™ Precast Gel.
2.Briefly rinse the gel cassette with ddH2O.
3.Remove tape and comb; avoid squeezing the gel.
4.Adapt Q-PAGE™ to electrophoresis system; instruction are provided below. (Invitrogen® Mini Gel Tank is recommended.)
5.Use a pipette to gently wash the wells with running buffer to remove residual storage buffer.
6.Fill the wells with running buffer prior to sample loading.
7.Load samples and pre-stained protein marker into numbered wells.
8.Fill both inner and outer chambers with running buffer to the highest level. Ensure gel wells are completely covered.
Power Setting for Running Q-PAGE™
Optimize the voltage and running time if needed.
| 150 V | 200 V*2 | 250 V*3 | 300 V*3 |
Running Time*1 | 50-70 mins | 35-55 mins | 25-40 mins | 15-30 mins |
Expected Current Initial (per gel) Final (per gel) |
35-45 mA 10-20 mA |
45-55 mA 20-25 mA |
75-85 mA 40-45 mA |
100-110 mA 60-70 mA |
Expected temperature | 25-30°C | 25-30 °C | 25-35°C | 30-40°C |
*1 Set voltage higher than 100 V is recommended.
*2 Try 200 V first, and optimize the voltage and running time if needed
*3 For higher voltage conditions, please use fresh running buffer for inner and outer chambers
*4 Running time varies depending on gel percentage, running buffer, temperature, and power supply.
Remove Q-PAGE™ Gel from Cassette
Open cassette immediately after electrophoresis. Avoid gel drying.
1.Insert the cassette opener into corners of cassette.
2.Sequentially pry the opener to separate the two plates.
3.Gently pull up notched plate and let gel stay on the front plate.
4.Use cassette opener to push through the slot in the cassette.
5.Carefully detach the gel from the bottom of gel.
- Avoid diagonally peeling the gel from the corner.
- If necessary, cut well separators with gel remover.
6.Gently remove the gel for further staining or Western blotting.
Gel Staining
Proteins separated using Q-PAGE™ Precast Gels can be further stained with most popular staining reagents, such as Coomassie dyes (R-250 or G-250), Silver-stain solution,
and FluoroStain™ Protein Fluorescent Staining Dye. (Cat. No. PS1000)
Transferring Protein from Q-PAGE™ to Blotting Membrane
1. After protein separation using Q-PAGE™, gently detach QPAGE™ from cassette and then equilibrate the gel in transfer buffer.
2. Pre-soak blotting membrane and filter papers in transfer buffer.
*Activate PVDF membrane in methanol before soaking in transfer buffer.
**Prepare 6 filter papers for one gel/membrane sandwich.
3. Assemble transfer sandwich by orientating cathode, sponge, filter papers, gel, membrane, filter papers, sponge, and anode. The protein goes to the direction of cathode to anode.
4. Carefully move roller over the gel/membrane to remove air bubbles and excess buffer until complete contact is established.
5. Insert transfer cassette into transfer module. Notice that black side of cassette should be next to black side of module.
6. Fill transfer tank with pre-cooled transfer buffer to the highest water level.
7. Set constant voltage at 100 V. Transfer for 90 minutes at low temperature condition. Pre-stained protein marker should be visible on the membrane after transfer is completed.
Transfer of proteins to the membrane can be checked using Ponceau S staining before blocking step.
Supplemental Information for Using Q-PAGE™ Precast Gel
Adapting Q-PAGE™ Midi Precast Gels to Invitrogen Mini Gel Tank Electrophoresis System
1. Place the Q-PAGE Midi Precast Gels with notched plate facing toward yourself. No extra adapter is needed.
2. Seat the gels on the bottom of Mini Gel Tank and close the cassette clamp.
3. Fill chambers with running buffer to the level of the fill line. Ensure gel wells are completely covered.
Adapting Q-PAGE™ Midi Precast Gels to other electrophoresis system, please follow the manufacturer’s instruction.
Buffer recipes
2X sample buffer with reducing agent
62.5 mM Tris-HCl pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)
10X Tris-Glycine running buffer
30.0 g Tris base, 144.0 g Glycine, 10.0 g SDS. Bring up the volume to 1 L with ddH2O.
1X running buffer
Dilute 100 ml 10X running buffer with 900 ml ddH2O.
10X transfer buffer
30.0 g Tris base, 144.0 g Glycine. Bring up the volume to 1 L with ddH2O.
1X transfer buffer
*Cool 1X transfer buffer to 4°C before using.
Dilute 100 ml 10X transfer buffer with 200 ml methanol and 700 ml ddH2O.
**Add SDS to 0.1% to promote transfer of high molecular weight proteins.
Troubleshooting Guidelines | ||
Problem | Possible Cause | Suggested Solution |
Well deformation | Pull one side of comb out of cassette. | Smoothly pull the comb straight out of the cassette. |
Bubbles between gel and cassette | Gel has been frozen or stored at wrong temperature. | Store Q-PAGE Precast Gels at 4°C. |
Buffer leaking from the inner chamber | Untight assembly of gels to the electrode modules | Reassemble Q-PAGE gels into the electrode modules. Fill outer chamber with 1X running buffer to the highest level. |
Samples do not sink into the wells. | Residual gel storage buffer in the wells | Rinse the gel wells with ddH2O or 1X running buffer before loading. |
Insufficient sample buffer | Use more sample buffer to prepare samples. | |
Current is zero and sample do not migrate into gel | Tape at bottom of gel not removed | Remove tape |
Gels run faster or more slowly than expected. | Incorrect running buffer | Check buffer composition. Use fresh 1X running buffer for inner chamber. |
Crooked bands at middle or bottom of gel | Gel has been frozen or stored at wrong temperature. | Store Q-PAGE Precast Gels at 4°C. |
Incorrect running buffer | Check buffer composition. Use fresh 1X running buffer for inner chamber. | |
Band pattern curves toward one or both sides of gel. | Buffer leaking from the inner chamber | Check assembly of gels into the electrode modules. |
Excessive heating of gel | Check buffer composition. Or dilute running buffer to 0.5-0.75X. Do not exceed recommended running conditions. | |
Insufficient buffer in inner or outer buffer chamber | Fill inner and outer chambers to completely cover gel wells. | |
Poor resolution or fuzzy bands | Excessive heating of gel | Check buffer composition. Do not exceed recommended running conditions. |
Incorrect running buffer | Check buffer composition. | |
Bands are missing on the membrane after Western transferring. | Proteins move in the wrong direction | Check the order of gel/membrane sandwich assembly, the direction of transfer cassette in transfer modules, and the polarity of connections to power supply. |
Swirls or missing bands; bands trail off in multiple directions on the membrane after Western transferring. | Contact between the membrane and the gel was poor; Air bubbles or excess buffer remains between the blotting membrane and the gel. | Use thicker/more filter paper in the gel/membrane sandwich Remove air bubbles and excess buffer between gel and membrane by carefully moving the roller over the membrane. |
Apparent molecular sizes of prestained protein markers are different as indicated. | Prestained protein markers used have not been calibrated for use with Q-PAGE gels. Dyes for staining protein markers affect the migration patterns of prestained proteins in different buffer systems. | Calibrate prestained protein markers against unstained proteins of known size or use SMOBIO’s ExcelBand™ Protein Markers. |
Q-PAGE™ Precast Gel
Gel Type | Bis-Tris | TGN (Tris-Glycine-Novel) | ||||||
Buffer systems | MOPS and MES | Tris-Glycine (Laemmli) | ||||||
Features | Clear and sharp bands, high resolution | Quick running, clear bands | ||||||
Cassette size | Mini Gel(10 x 8.3 cm) | Midi Gel(10 X 10 cm) | Mini Gel(10 x 8.3 cm) | Midi Gel(10 X 10 cm) | ||||
Electrophoresis system | Bio-Rad systems | Mini Gel Tank Xcell SureLock, Hoefer SE260 | Bio-Rad systems | Mini Gel Tank Xcell SureLock, Hoefer SE260 | ||||
Well format & Capacity | 12 wells, 25 μl/well | 15 wells, 22 μl/well | 12 wells, 40 μl/well | 15 wells, 28 μl/well | 12 wells, 25 μl/well | 15 wells, 22 μl/well | 12 wells, 40 μl/well | 15 wells,28 μl/well |
Gel percentage/ Cat. No. | 8% | 8% | 8% | 8% | 10% | 10% | 10% | 10% |
QP2110 | QP2120 | QP3110 | QP3120 | QP4210 | QP4220 | QP5210 | QP5220 | |
12% | 12% | 12% | 12% | 4-15% | 4-15% | 4-15% | 4-15% | |
QP2310 | QP2320 | QP3310 | QP3320 | QP4510 | QP4520 | QP5510 | QP5520 | |
4-12% | 4-12% | 4-12% | 4-12% |
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QP2510 | QP2520 | QP3510 | QP3520 |
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ExcelBand™ Protein Markers
Ready-to-use— premixed with a loading buffer for direct loading, no need to boil
Broad range— 310 kDa to 5 kDa
Pre-stained bands — for monitoring protein separation during electrophoresis and Western blotting transferring efficiency on membrane
Enhanced bands— for quick reference
YesBlot™ Western Marker I
Ready-to-use — no need of mixing or heating before sample loading
Direct visualization — 10 IgG-binding proteins for direct visualization on Western blots
Pre-stained bands — 4 pre-stained proteins for monitoring protein separation during electrophoresis and Western blotting transferring efficiency on membrane
Wide range — 10 clear bands from 15 to 200 kDa for size estimation
Quick reference — two enhanced bands (30 and 80 kDa)
FluoroStain™ Protein Fluorescent Staining Dye
Compatible to MASS analysis — compatible to the analysis of mass spectra, such as LC-MS/MS, MALDI-TOF, and etc.
High sensitivity — detection level achieve ~3 ng, similar to silver staining
Substitution of the Coomassie Blue protein staining method
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链接很容易，就用EDC或者CMC等碳二亚胺，是最容易在羧基和氨基之间催化形成酰胺键，反应条件就是0到4度，过夜，用磁力搅拌子温和搅拌，pH<7反应快，不过pH=7或稍微pH>7也关系不大。EDC或者CMC等碳二亚胺催化活性极高，而且一般的蛋白质的分子表面总有Lys,Arg,Asn,Gln，所以游离在分子表面的羧基和氨基总是不少的，参与反应的两种蛋白质用大致10：1的比例混合（哪种蛋白质便宜，哪种蛋白质的物质的量就大些，但是这不是全部原因），反应样品浓度就按照平时使用时的浓度或略微更低浓度（别用储存液的浓度）即可。很容易反应，而且EDC或者CMC等碳二亚胺是零臂连接试剂，用于空间位阻，形成多分子交联可能较小，即使形成了也很容易用分子筛层析按照分子量区分开。
参与反应的两种蛋白质用大致10：1的比例混合（哪种蛋白质便宜，哪种蛋白质的物质的量就大些，但是这不是全部原因），为了避免两种蛋白质之间发生交联成为串联，因为一种蛋白质形成串联的可能性低一些；即使要形成串联的蛋白质串珠，最好也不要是价格高的那种；另外，EDC或者CMC等碳二亚胺催化活性极高，室温下即会催化，所以要在0到4度，过夜，用磁力搅拌子温和搅拌，一方面避免碳二亚胺催化活性太高形成串联的蛋白质串珠，另一方面，保护蛋白质不变性；反应样品浓度就按照平时使用时的浓度或略微更低浓度（别用储存液的浓度）即可，为什么？因为浓度高了更易形成串联的蛋白质串珠结构，浓度低了，没有反应成功，通过分子筛层析还可以分离出来接着连接，如果形成了酰胺键再要专一性切开就没有那么容易了。
1） 物素N—羟基丁二酰亚胺酯（biotinyl-N-hydroxy-succinnimideBNHS）制备：
取物素1g混悬于12 mlN,N-二甲基甲酰胺(DMF)加0．6g N-羟基丁二酰亚胺(HOSU)0. 8 g二环基碳二亚胺(DCC)置于密闭容器内室温磁力搅拌作用夜滤滤液经旋转蒸干加10mL乙醚洗涤继加200 ml异丙醇使其重结晶获白色粉末状晶体
BNHS酯键-C=0基团与蛋白质赖氨酸氨基结合使物素标记蛋白含赖氨酸残基越或蛋白质等电点pI 6.0其标记效越BNHS适用于标记抗体及性偏碱性抗原
【过敏性咳嗽血清学IGE检查】
以过敏患者血清作为实验材料的本外试验方法称为血清学试验。其它体液如炎症部位分泌物、渗出物、灌洗液也可采用相同的实验方法进行检测。主要检测项目有总IgE和特异性IgE，即过敏原特异性IgE。
【什么是总IgE？】
IgE即免疫球蛋白E，是I型变态反应病如过敏性鼻炎、过敏性哮喘、异位性皮炎、湿疹、急慢性荨麻疹发病机制中起主要作用的免疫分子，因而在过敏反应的免疫学实验诊断中是首选的检测项目。总IgE是过敏性疾病的特异性检查项目，IgE水平增高提示I型变态反应病的可能性大，但不能用于判断过敏原。
【IgE的特点】
IgE是血清浓度最低的免疫球蛋白 ，只有血清中IgG浓度的万分之一。IgE对热不稳定，是半衰期最短一的免疫球蛋白 ，只有2.8天，与细胞表面结合的IgE半衰期稍长，8~14天，IgE由变应原入侵部位（鼻咽、支气管、胃肠道）的黏膜固有层中的浆细胞合成。在各类免疫球蛋白中，IgE是合成率最低、分解率最高的。属于亲细胞抗体，过敏体质者的胎儿脐带血中IgE浓度可能升高，检测脐血中IgE浓度可用于评估胎儿过敏体质的可能性。
【IgE检测方法】
通常用ELISA方法检测总IgE。由于血清IgE浓度很低，一般酶免疫试验方法的敏感性不足以检出血清IgE，现在常规实验室检测血清IgE的试剂盒采用生物素——抗生物素蛋白 放大的ELISA。试剂盒中所含用于制定标准曲线的IgE标准品和检测结果的IgE浓度单位与其它免疫球蛋白 不同，不是用mg/L表示，而是用u/ml或ku/l表示。
【IgE的正常值（参考范围）】：
血清IgE水平在正常人群中呈偏态分布，即多数人为0或接近于0，IgE水平越高的人数越少。因此计算平均值时应计算几何平均值才能反映其真实情况，即用对数转换后其分布才能近似正态分布。
健康人群血清IgE水平与年龄关系较大，小儿和老年人的IgE水平低于成年人。新生儿血清中IgE水平很低，接近于零。随年龄增长，IgE水平也不断升高，5~7岁后接近正常人水平。按Pharmacia公司提供的参考范围，1个月以内<12KU/L,1岁<11KU/L,2~4岁<33KU/L,5岁以上至成人<85KU/L.
过敏性疾病患者的血清IgE水平可达2000~8000KU/L,当IgE水平高于2000KU/L时应考虑寄生虫感染.
有时血清总IgE水平检测结果为0或参考范围内低值,并不能排除过敏性疾病的可能,须结合临床表现和血清特异性IgE检测结果进行判断.
【什么是特异性IgE检测(sIgE）？】
通常所称的过敏原检测，并非真正检测血液样本中的过敏原分子，而是间接地检测其中针对某种过敏原的特异性IgE分子，特异性IgE检测实际上是检测过敏原特异性IgE，即检测样本中针对某种变应原的特异性IgE，从而间接地判断患者是否对某种过敏原过敏。
环境中常见的过敏原包括以下类别：
寄生虫和微生物：各种螨类（屋尘螨和粉尘螨等）、各种真菌（点青霉、烟曲霉、分枝孢霉、交连孢霉等）、蟑螂。
植物花粉：各种草花粉（豚草、葎草、蒿草）、各种树花粉（桑树、柏树、悬铃木、桦树、榆树、柳树、杨树等）。
动物皮毛：猫、狗、马、鸽子等动物的毛和皮屑。
叶酸和生物素都属于B族维生素,
对维持机体的生理功能有重要作用。
求速度考试，简答
