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[Bio101] Laemmli-SDS-PAGE
[Bio101] Laemmli - SDS - PAGE电泳   

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Abstract

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate proteins with relative molecular mass no smaller than 10 KD. Very small proteins (<10 KD) are difficult to resolve due to low ability of binding to SDS, which can be solved by gradient gels or using different eletrophoresis conditions, like Tricine-SDS-page. The basic Laemmli SDS PAGE procedure is described here.

Keywords: SDS-PAGE(SDS-PAGE), Protein(蛋白质), Electrophoresis(电泳)

Materials and Reagents

  1. Pre-stain Protein MW marker (Bio-Rad Laboratories)
  2. TEMED (Life Technologies, Gibco®)
  3. Ammonium persulfate (Sigma-Aldrich)
  4. SDS (Research Organics INC)
  5. 30% Acrylamide stock (37.5: 1 acrylamide: bisacrylamide) (Bio-Rad Laboratories)
  6. Bromophenol Blue (Thermo Fisher Scientific)
  7. Tris Base (Calbiochem-Behring)
  8. Glycine (EM Science)
  9. EDTA (Amresco)
  10. Glycerol (EM Science)
  11. Isopropanol
  12. Tris-HCl (pH 6.8)
  13. β-mercaptoethanol (Sigma-Aldrich)
  14. 10x running buffer (see Recipes)
  15. 2x SDS protein sample buffer (see Recipes)

Equipment

  1. Protein mini gel cassettes (Bio-Rad)
  2. Heating block module
  3. Table-top centrifuge
  4. Power supply
  5. Gloves
  6. Filter paper

Procedure

  1. Making SDS-PAGE gel
    1. Clean and completely dry glass plates, combs, and spacers are required.
    2. Assemble gel cassette by following manufacturer instructions.
    3. Prepare 10% lower gel (separating gel) by adding the following solutions (wear gloves when prepare gel solution) (total volume= 5 ml)
      2 ml ddH2O
      1.67 ml 30% acrylamide/Bis
      1.25 ml 1.5 M Tris (pH 8.8)
      25 μl 20% SDS
      25 μl 10% ammonium persulfate (make it fresh and store at 4 °C up to a month)
      2.5 μl TEMED (add it right before pour the gel)
      Note: Change ration of ddH2O to 30% acrylamide/Bis to get different percentage of separating gel.
    4. To avoid polymerization, after adding TEMED, mix well and quickly transfer the gel solution by using 1 ml pipette to the casting chamber between the glass plates and fill up to about 0.7 cm below the bottom of comb when the comb is in place.
    5. Add a small layer of isopropanol to the top of the gel prior to polymerization to straighten the level of the gel.
    6. Once the gel has polymerized, start to prepare stacking gel (5%) by adding the following solutions (total volume= 3 ml)
      2.088 ml dH2O
      0.506 ml 30% acrylamide/Bis
      0.375 ml 1 M Tris (pH 6.8)
      15 μl 20% (w/v) SDS
      15 μl 10% ammonium persulfate
      1.5 μl TEMED (add it right before the gel is poured)
    7. Remove the isopropanol layer by using filter paper. Rinse the top layer of the gel with ddH2O and dry off as much of the water as possible by using filter paper.
    8. Add TEMED and mix the stacking gel solution well. Quickly transfer the gel solution by using a 1 ml pipette till the space is full, and then insert the appropriate comb.
    9. Allow the top portion to solidify and then carefully remove the comb.
      Note: The gels can be stored with the combs in place tightly wrapped in plastic wrap and put in a second container with wet tissue towel (keep the gels moist) at 4 °C for 1 to 2 weeks.

  2. Sample preparation
    1. Prepare same amount of protein samples according to BCA assay result, see BCA (bicinchoninic acid) protein assay.
    2. Add the same volume of 2x protein sample buffer to each protein sample, mix and boil the samples at 95 °C heating block module for 10 min.
    3. Spin the samples at the maximal speed for 1 min (samples from some tissue/cell sources may need longer spin) in tabletop centrifuge and leave the samples at room temperature until you are ready to load onto the gel.
      Note: Can store extracted protein samples (containing sample buffer) at -20 °C and reheat at 95 °C for 5 min when used the following time.

  3. Electrophoresis
    1. Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate on the inside. Press down on the electrode assembly while clamping the frame to secure the electrode assembly and put the clamping frame into the electrophoresis tank.
    2. Pour some 1x electrophoresis running buffer into the opening of the casting frame between the gel cassettes. Add enough buffer to fill the wells of the gel. Fill the region outside of the frame with 1x running buffer.
    3. Slowly load the same amount of protein samples into each well as well as load 10 μl of protein MW marker.
    4. Connect the electrophoresis tank to the power supply.

  4. Protein detection
    1. If protein of interest is about 0.2 μg or more in the sample, typically use Coomassie blue staining (see Coomassie blue staining). Otherwise, use silver staining (sliver staining), which is more sensitive and can detect as little as 5 ng protein.

Recipes

  1. 10x running buffer
    30.3 g Tris-base
    144.0 g glycine
    10.0 g SDS
    Completely dissolve in about 800 ml ddH2O and then more ddH2O up to 1 liter.
  2. 2x SDS protein sample buffer
    1.25 ml 1 M Tris-HCl (pH 6.8)
    4.0 ml 10% (w/v) SDS
    2.0 ml glycerol
    0.5 ml 0.5 M EDTA
    4 mg bromophenol blue
    0.2 ml b-mercaptoethanol (14.3 M)
    Bring the volume to 10 ml with ddH2O.

References

  1. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.

简介

十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)用于分离相对分子量不小于10KD的蛋白质。 由于与SDS的结合能力低,非常小的蛋白质(<10KD)难以解析,这可以通过梯度凝胶或使用不同的电泳条件(如Tricine-SDS-PAGE)来解决。 这里描述了基本的Laemmli SDS PAGE方法。

关键字:SDS-PAGE, 蛋白质, 电泳

材料和试剂

  1. 预染色蛋白MW标记(Bio-Rad Laboratories)
  2. TEMED(Life Technologies,Gibco )
  3. 过硫酸铵(Sigma-Aldrich)
  4. SDS(Research Organics INC)
  5. 30%丙烯酰胺储备液(37.5:1丙烯酰胺:双丙烯酰胺)(Bio-Rad Laboratories)
  6. 溴苯酚蓝(Thermo Fisher Scientific)
  7. Tris碱(Calbiochem-Behring)
  8. 甘氨酸(EM Science)
  9. EDTA(Amresco)
  10. 甘油(EM Science)
  11. 异丙醇
  12. Tris-HCl(pH 6.8)
  13. β-巯基乙醇(Sigma-Aldrich)
  14. 10x运行缓冲区(参见配方)
  15. 2x SDS蛋白样品缓冲液(见配方)

设备

  1. 蛋白质微型凝胶盒(Bio-Rad)
  2. 加热块模块
  3. 台式离心机
  4. 电源
  5. 手套
  6. 过滤纸

程序

  1. 制作SDS-PAGE凝胶
    1. 需要清洁和完全干燥的玻璃板,梳子和垫片。
    2. 按照制造商的说明装配凝胶盒
    3. 通过加入以下溶液(当制备凝胶溶液时戴手套)制备10%下凝胶(分离凝胶)(总体积= 5ml)
      2ml ddH 2 O 2 / 1.67ml 30%丙烯酰胺/Bis 1.25ml 1.5M Tris(pH8.8)
      25μl20%SDS
      25μl10%过硫酸铵(使其新鲜,在4°C储存一个月)
      2.5μlTEMED(在倒入凝胶之前加入)
      注意:将ddH 2 O的比例改为30%丙烯酰胺/Bis以获得不同百分比的分离凝胶。
    4. 为了避免聚合,在添加TEMED后,充分混合并通过使用1ml移液管将凝胶溶液快速转移到玻璃板之间的浇铸室,并且当梳子就位时填充到梳子底部以下约0.7cm。
    5. 在聚合之前向凝胶顶部添加一小层异丙醇以使凝胶水平变直
    6. 一旦凝胶聚合,通过加入以下溶液(总体积= 3ml)开始制备堆叠凝胶(5%)。
      2.088ml dH 2 O 2 / 0.506ml 30%丙烯酰胺/Bis 0.375ml 1M Tris(pH 6.8)
      15μl20%(w/v)SDS
      15μl10%过硫酸铵
      1.5μlTEMED(在凝胶倒入之前加入)
    7. 使用滤纸去除异丙醇层。 用ddH 2 O 2冲洗凝胶的顶层并使用滤纸尽可能多地干燥水。
    8. 加入TEMED并混合堆叠胶体溶液。 通过使用1毫升移液管快速转移凝胶溶液,直到空间充满,然后插入适当的梳子
    9. 让顶部固化,然后小心地取出梳子。
      注意:凝胶可以与梳子一起保存在紧密包裹在塑料包装中的位置,并置于具有湿纸巾毛巾(保持凝胶湿润)的第二个容器中,在4℃下1至2周。

  2. 样品制备
    1. 根据BCA测定结果制备相同量的蛋白质样品,参见BCA(二金鸡宁酸)蛋白质测定。
    2. 向每个蛋白质样品中加入相同体积的2x蛋白质样品缓冲液,在95℃加热块模块中混合并煮沸样品10分钟。
    3. 在台式离心机中以最大速度旋转样品1分钟(来自一些组织/细胞来源的样品可能需要更长时间旋转),并将样品保持在室温,直到准备装载到凝胶上。
      注意:可以将提取的蛋白质样品(含有样品缓冲液)储存在-20°C,并在95°C再加热5分钟,然后在以下时间使用。

  3. 电泳
    1. 从铸造台上取下凝胶盒,将其放在电极组件中,短板在内侧。向下压电极组件,同时夹紧框架以固定电极组件,并将夹紧框架放入电泳槽
    2. 将一些1x电泳缓冲液倒入凝胶盒之间的铸造框架的开口。加入足够的缓冲液以填充凝胶的孔。用1x运行缓冲区填充框架外部的区域。
    3. 缓慢加载相同量的蛋白质样品到每个孔以及加载10微升蛋白质MW标记
    4. 将电泳槽连接到电源

  4. 蛋白检测
    1. 如果样品中感兴趣的蛋白质为约0.2μg或更多,通常使用考马斯蓝染色(参见考马斯蓝染色)。 否则,使用银染(条纹染色),这是更灵敏,可以检测低至5 ng蛋白质。

食谱

  1. 10x运行缓冲区
    30.3克Tris-碱
    144.0g甘氨酸
    10.0克SDS
    在约800ml ddH 2 O中完全溶解,然后在1升内更多的ddH 2 O 2。
  2. 2x SDS蛋白样品缓冲液
    1.25ml 1M Tris-HCl(pH6.8)
    4.0ml 10%(w/v)SDS 2.0ml甘油 0.5ml 0.5M EDTA
    4mg溴酚蓝
    0.2ml b-巯基乙醇(14.3M) 用ddH 2 O将体积调至10ml。

参考文献

  1. Laemmli,U.K。(1970)。 在噬菌体T4头部装配过程中切割结构蛋白。 自然 227(5259):680-685。
  • English
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol Bio101: e80. DOI: 10.21769/BioProtoc.80;
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rajendran nair
mahatmagandhicollege
Lane A Lane B Lane C Lane D Lane E Lane F Lane G
No 1 172,847.185 172,847.185 166,580.317 205,000.000 142,554.536 142,554.536
No 2 109,761.954 109,761.954 109,761.954 97,400.000 95,218.686 94,595.384
No 3 97,088.395 97,088.395 97,088.395 66,000.000 72,348.988 79,908.075
No 4 92,725.123 92,725.123 92,413.345 43,000.000 43,000.000 43,000.000
No 5 85,233.211 85,233.211 85,545.881 29,000.000 37,920.673
No 6 76,135.568 76,450.447 31,694.655
No 7 49,137.611 49,137.611 21,114.943
No 8 6,149.425



Lane A – ASC Puntius
Lane B – ASC Etroplus
Lane C –PDC Puntius
Lane D-PDC Etroplus
Lane E- Protein molecular weight marker 29 -205 KDa (Genei,Bangalore)
Lane F-Protein in Etroplus
Lane G- Protein in Puntius

please let me know how I can interpret the documentation of protein and collagen of the fishes puntius and etroplus

12/23/2014 8:32:54 AM Reply
chiging yadii chiging
CIFE
For running SDS for collagen, how do i prepare my sample? i have extracted collagen in lyophillized form. do i have to directly use it as my sample or should prepare by dissolving in acids. and if it is, kindly tell me the ratio and molarity.
6/19/2014 12:36:15 AM Reply
Fanglian He
University of Pennsylvania

Hi, Sorry I do not have any experience with running collagen on a SDS-page gel. But, I have passed your request to Bio-protocol Editorial Committee. They will recommend/invite a research group to contribute the requested protocol to Bio-protocol in the near future. Will keep you posted on this.

--Fanglian

6/23/2014 10:03:25 AM


Kaidu Barrosa
Unifesp

Hello! I'm also interested in collagen identification through SDS-page!Could you please recommend me such reference as well, if you please? Thank you so much!

Kaidu Hanashiro Barrosa,
kaiduhb@gmail.com

12/11/2014 10:06:08 AM


asif ahmed
university of greenwich

hi dear.
How can i prepare the sample buffer of 20 ml for the yeast secreted proteins seperation. Please mention the percentage of each components. Can I add B-mercaptoethanol during sample buffer preparation or I need to add during sample preparation?. thanks

12/18/2014 5:08:28 AM


Katerine Carreño
CICESE

Hi Fanglian, I'm interested in collagen separation on a SDS-PAGE gel too! Can you please pass my request to Bio-protocol Editorial Committee? I'll appreciate it so much!

Best regards,
Katerine Carreño
katerine.carreno@gmail.com

1/30/2015 5:47:25 PM


Katerine Carreño
CICESE

Hi again, Sorry, I wrote the wrong email, it is katica.carreno@gmail.com.

Thank you!

Katerine Carreño

1/30/2015 5:48:51 PM


Geneva Cruz
Temple University

Did they ever get a Collagen protocol made? I am interested in one as I am working with Collagen I currently. Thanks

2/15/2017 2:59:56 PM


Fatemeh Rostami
Isfahan University
Hi I can introduce my paper and my extraction buffer which I made and applied for leaf samples for SDS PAGE:

Rostami, F and Ehsanpour, A. A. 2009. Application of silver thiosulfate (STS) on silver accumulation and protein pattern of potato (Solanum tuberosum L.) under in virto culture. Malaysian Applied Biology Journal. 32(2), 49-54.
4/29/2014 4:13:47 AM Reply
rogini sivalingam
universiti malaysia kelantan
Hi, can u suggest me the proper extraction buffer for leaf samples for coomassive briliant blue staining for sds page method. Can u also give me instruction how to prepare the extraction buffer.
4/25/2014 11:16:50 PM Reply
Fanglian He
University of Pennsylvania

I am not sure what kind of plant leaf you are interested in.

For Arabidopsis leaf, you may check out the following protocol.

Flury et al. (2013). MAPK Phosphorylation Assay with Leaf Disks of Arabidopsis. Bio-protocol 3(19):http://www.bio-protocol.org/wenzhang.aspx?id=929

For tobacco leaf,
Husk et al. (2014). Monoclonal Antibody Purification (Nicotiana benthamiana Plants).Bio-protocol 4(2):http://www.bio-protocol.org/wenzhang.aspx?id=1034

Both above protocols have details about how to extract proteins from leaves including the recipes of the extraction buffers.Hope you would find them helpful.

4/28/2014 3:54:41 PM


Romen Naorem
Dibrugarh University
Why do we use different PH in stacking gel and separating gel in SDS PAGE?
What is the main role of glycerol/ glycerin in running buffer?
2/24/2014 10:06:35 PM Reply
Fanglian He
University of Pennsylvania

Laemmli-SDS-PAGE system is discontinuous buffer system. Different PH (and ionic strength) in stacking gel and separating gel can help protein samples concentrate/stack in stacking gel before running in a separating gel. Thus, the resolution of discontinuous buffer system much better than that of continuous systems (in particular when the volume of a protein sample is large).

Running buffer contains glycerine, not glycerol. Glycerine is a weak acid used to adjust the pH of the running buffer.

Glycerol in protein sample buffer is heavier (more dense) than water so that it makes protein sample sink at the bottom of the sample well instead of flowing around in the upper running buffer.


4/28/2014 3:29:47 PM


Romen Naorem
Dibrugarh University

I performed SDS-PAGE by using total bacterial protein samples obtained by sonicaition method having volume of 40 micro litre and protein marker of 20 micro litre. But in this experiment I am not able to get the protein bands after destaining process. I like to know is what is possible trouble shooting in my experiment.

5/2/2014 11:08:09 PM


cecilia ojesola
Federal university of agriculture, abeokuta
how do i interprete the gel after destaining, i mean the bands
8/20/2013 6:54:12 AM Reply
Fanglian He
University of Pennsylvania

I am not quite sure what your question is. In general, while looking at your gel, the first question is:
where is the band for your protein of interest (POI)?
--Based on the sizes of makers and known MW of POI, you should know where the band of POI is expected to be.
--If the expression level of POI is very low, you may try some sensitive staining (like silver staining)
--I'd also mention that one single band on SDS-PAGE could consist of more than one protein, which have similar MW. You can do 2D-gel to further separate it.
--It would be very helpful if you have controls to confirm your results. Best way to confirm you result is to do Western Blot using ABs against POI.

Hope I have answered your question.

8/20/2013 9:57:20 AM


Can we determine the protein purity with SDS-PAGE? for our target protein of course.
I will be grateful if you can help me with this.
2/21/2013 11:05:40 AM Reply
Mohammed Amin
LNCPP
why filgrastim is analyzed with sds-page and not western blot ?
2/19/2013 11:04:40 AM Reply
chris adhiyanto
Universitas Islam Negeri Jakarta Indonesia
Dear

I want to analyze ankyrin membrane erythrocyte by SDS-Page. do you know, where the best condition for this protocol? I has been tried separate by gradient method (Laemmli and Fairbanks), but the result was not satisfied. And when I used 12.5% SDS Page, I found the ankyrin band, but the position is near with spectrin. Please if some one know the best method, please give me suggestion.
8/29/2012 5:53:23 PM Reply
Fanglian He
University of Pennsylvania

Sorry I do not know the answer to your question. But, I will check it around and keep you posted.

8/31/2012 12:17:54 PM


周 桐
北京化工大学
3/19/2012 5:19:36 PM Reply
Fanglian He
University of Pennsylvania

Yes, this is a protocol for the classic SDS-PAGE system, which was developed by Laemmli.

3/21/2012 1:37:15 AM