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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, Protein, Electrophoresis

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)


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


  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.


  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.


  1. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2011). Laemmli-SDS-PAGE. Bio-101: e80. DOI: 10.21769/BioProtoc.80.
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Karnchita Thitaprom
Kasetsart University
Hello Dear.. I am interested in determining a protein profile of Tempe by Glycine SDS-PAGE
I found paper ' Characterization of biochemical and functional properties of water-souble Tempe flour' which it that recommended Laemmli method. I am so confused about preparing sample buffer. Buffer prepares that mixing between 0.01M Tris-HCl, pH 6.8, 0.1% SDS and 0.1% 2-mercaptoethanol . But they didn't write about ratio of the above solution
And Can I use this protocal to determine

May you suggest me about this issue , Thank a lot Dear

E-mail :
6/9/2019 9:01:17 PM Reply
farhan nabilah
UiTM Shah Alam
Hello...I am also interested in determining the collagen and gelatin identification through SDS PAGE. Could u recommend me some method frm Bio-protocol. I appreciate it so much!!!
5/15/2019 1:04:07 AM Reply
Courtney Broady
University of Tennessee
Hello, I am working out the logistics for running an SDS PAGE gel to look for apolipoprotein B-100 which is 550kDa. As for the volume of protein, I need to mix with the sample buffer and the molecular weight marker are they all the same? Also because the sample buffer is stained, is it still necessary to use a protein mw marker for each well? Could it only be loaded into the first well?
6/13/2018 7:35:52 AM Reply
rajendran nair
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
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

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.


6/23/2014 10:03:25 AM Reply

Kaidu Barrosa

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,

12/11/2014 10:06:08 AM Reply

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 Reply

Katerine Carreño

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

1/30/2015 5:47:25 PM Reply

Katerine Carreño

Hi again, Sorry, I wrote the wrong email, it is

Thank you!

Katerine Carreño

1/30/2015 5:48:51 PM Reply

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 Reply

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

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):

For tobacco leaf,
Husk et al. (2014). Monoclonal Antibody Purification (Nicotiana benthamiana Plants).Bio-protocol 4(2):

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 Reply

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

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 Reply

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 Reply

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

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 Reply

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
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

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

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 Reply

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