Navigate this Article


Very Low Molecular Weight Proteins Electrophoresis Protocol Updated   

How to cite Favorites 4 Q&A Share your feedback Cited by


The electrophoresis is the most used technique to separate proteins and usually the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proposed by Laemmli is the prefered since it can diferentiate large size proteins, but for very low molecular weight proteins such as 3 KDa and below becomes difficult. Thus a modification of the basic Laemmli SDS-PAGE protocol was required to allow for proteins up to 10 KDa to be separated, maintaining good resolution and reproducible results. This work demonstrates how a 18% gel and modifications of the basic Laemmli protocol acrylamide gel let protein samples with 1 KDa and 0.6 KDa be visible and separated.

Keywords: Electrophoresis, SDS-PAGE, Proteins, Low Molecular Weight, Separation, Purification


Electrophoresis is a technique in which using an electric field separates molecules. “A very common electrophoresis method to separate and to denature proteins which uses a discontinuous polyacrylamide gel as a support medium is called Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The most commonly used protocol is also called the Laemmli method which refers to the first published protocol of this technique established by Laemmli in 1970 (Ornstein, 1964).

The SDS-PAGE can be used to separate proteins, estimate relative molecular mass, determine the relative abundance of major proteins in a sample, and/or determine the distribution of proteins among fractions. Different staining methods can be used to detect rare proteins and to address their biochemical properties. Rath in her work compares the migration of reference proteins at different gel concentration with gels ranging from 11 to 18% not getting a good resolution in molecules under ~6 KDa (Rath et al., 2013; DeWald et al., 1986).

In order to analyze low molecular weight proteins by this electrophoresis system, high concentration gels are needed, but these gels are very brittle and thus difficult to handle. With a modification of the basic Laemmli SDS-PAGE protocol, proteins with 10 KDa or less will be separated maintaining a good resolution with reproducible results. This method will be explained in this work and demonstrated using 1 KDa and 0.6 KDa proteins as samples.

Materials and Reagents

  1. Glass plate
  2. Pre-stained Protein MW marker (Amersham, catalog number: RPU755)
  3. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678)
  4. SDS (Sigma-Aldrich, catalog number: L3771)
  5. Acrylamide-bis (19:1) (Bio-Rad Laboratories, catalog number: 161-0123)
  6. Bromophenol Blue (Sigma-Aldrich, catalog number: B0126)
  7. Tris-base (Promega, catalog number: H5131)
  8. Glycine (Bio-Rad Laboratories, catalog number: 161-0718)
  9. EDTA (Sigma-Aldrich, catalog number: E9884)
  10. Glycerol (Promega, catalog number: H5433)
  11. TEMED (HiMedia, catalog number: RM1572)
  12. Urea (MP Biomedicals, catalog number: 103209)
  13. HCl 37% (Riedel de Haen, catalog number, 30721-2.5L-GL)
  14. Resolution Buffer (Lower) 4x (see Recipes)
  15. Stacking Buffer (Upper) 4x (see Recipes)
  16. 10x Running buffer (see Recipes)
  17. Sample buffer (see Recipes)


  1. 1 ml pipette
  2. Protein mini gel cassettes (Bio-Rad Laboratories, catalog number: 1658000FC)
  3. Power Supply (Bio-Rad Laboratories, catalog number: 1645070)


  1. For the Gel preparation after cleaning the glass plates, assemble the cassettes following the manufacturer’s instructions. Using the recipe below (Table 1) the two gels are mixed, first the separation gel according to the order described and then the stacking gel. To avoid polymerization, after adding TEMED, mix well and quickly transfer the gel solution by using a 1 ml pipette to the casting chamber between the glass plates and fill up to about 6 cm from bottom to top. Then, prior to polymerization add dH2O to straighten the level of the gel. After polymerization remove the water, prepare the stacking gel and follow the same steps, prior to polymerization insert the appropriate comb until polymerization.

    Table 1. 18% Polyacrylamide gel recipe for very low molecular weight protein

    As soon as the gel is polymerized it can be mounted into an electrophoresis tank, and then filled with running buffer between the gel cassettes and the outside region. 
  2. Once the electrophoresis tank is connected to the power supply, perform a pre-run for 15 min at 20 A. After the pre-run, load the prepared samples into the different wells of the gel, approximately 10 µl of the samples are loaded with sample buffer (5 µl of sample and 5 µl of sample buffer without heating) and 5 µl of the MW marker. The run will be held for 45 min under constant current at 25 A. 
  3. The samples used here are microcystins, a hepatotoxin produced by cyanobacteria with a theoretical weight of 0.995 KDa and Bromophenol blue with a theoretical weight of 0.669 KDa (Reference 3). After the run, stain the gel with Coomassie Blue. The resulting run (Figure 1) shows a clear difference between the microcystin samples and the bromophenol blue marker.
  4. If the mass of protein of interest present in the sample is about 0.2 μg or more, the Coomassie blue staining is typically used. Otherwise, silver staining can be an alternative, as it is more sensitive and can detect masses of proteins as little as 5 ng.

    Figure 1. Polyacrylamide gel. On lane 1 molecular weight marker, lane 2 Bromophenol Blue, lane 3 white, lane 4 sample 1, lane 5 sample 2.


  1. Resolution Buffer (Lower) 4x
    181.71 g (1.5 M) (Adjust pH to 8.8 with HCl)
    4 g (0.4%)
    c.s.p.1 L
  2. Stacking Buffer (Upper) 4x
    60.58 g (0.5 M) (Adjust pH to 6.8 with HCl)
    4 g (0.4%)
    c.s.p.1 L
  3. Running Buffer 10x
    1.92 M
    0.25 M
    SDS 1%
  4. Protein samples Buffer 4x
    200 mM, pH 6.8
    EDTA  400 mM
    SDS 8%
    Glycerol 40%


This work was possible thanks to the IDEA foundation, the ecology and aquatic system’s laboratory from the Venezuela´s Central University, and is a modification of Laemmli (1970).
  We wish to thank the anonymous reviewers and to Carlos Cáceres and Ysvic Inojosa for his comments and corrections on the manuscript.

Competing interests

The author declares no conflict of interest.


  1. DeWald, D. B., Adams, L. D. and Pearson, J. D. (1986). A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000. Anal Biochem 154(2): 502-508.
  2. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
  3. National Center for Biotechnology Information. PubChem Compound Database; CID=8272, (accessed Sep. 25, 2018).
  4. Ornstein, L. (1964). Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci 121: 321-349.
  5. Rath, A., Cunningham, F. and Deber, C. M. (2013). Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts. Proc Natl Acad Sci U S A 110(39): 15668-15673.
Please login or register for free to view full text
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Rivera, C. E., Rosales, J. D., Freites-Perez, J. C. and Rodriguez, E. (2018). Very Low Molecular Weight Proteins Electrophoresis Protocol. Bio-101: e3093. DOI: 10.21769/BioProtoc.3093.
By submitting a question/comment you agree to abide by our Terms of Service. If you find something abusive or that does not comply with our terms please contact us at

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.

vu hung
Immanuel Kant Baltic Federal University
Hello, thank you for your protocol. I'm interested in peptide 2 kDa. I have tried your protocol: 18% glycine - SDS- PAGE, 25 A for 3,5 hours. I use ruler 10- 250 kDa. The green band is 10kDa. And this is the results. Nearby the green band is 2 lanes of bromophenol (0,6 kDa as you wrote). I have 2 question:
- How can i reduce the time, because 3,5 hours is too long,
- How can i separate the geen band and bromophenol so that i can sure that i can detect peptide 2kDa between them.
Can i make 25% gel and put more ure??? Please reply me!!!! Thanks a lot
10/20/2020 6:48:52 AM Reply
jose rosales

Good morning, in the gel I see that the green band is very close to the end of the gel; which could indicate that the low molecular weight band came out of the gel during the electrophoretic run.

In the gel that I show the 10 kDa band is in the middle of the gel. Please check the acrylamide concentration of your gel.

You can use 20% acrylamide and 20 minutes of pre-run.

It does not change the urea concentration.

The amperage can be 30 mAmp, but take care of the temperature, that the buffer does not overheat, a cold room is useful.

10/21/2020 5:29:30 AM Reply

vu hung
Immanuel Kant Baltic Federal University

Thank you for reply, can i ask 2 more questions:
- Why do we need to pre-run?
- in most of protocols, we need to run with samples at low Voltage so that protein can concentrate in stacking gel --- but don't we need this step for low molecular weight proteins???
Best regards,

10/22/2020 9:54:44 PM Reply

Shammi Akter
Pohang University of Science and Technology (POSTECH)
Hi, Thanks for reply. I have one more question about Very Low Molecular Weight Proteins Electroscopes Protocol. When I added 3 gm urea following this recipe (Separating gel) I could not add 2 ml water, because of volume increasing. Please give me a suggestion how can i solve this problem. Thank you.
7/16/2020 8:18:37 PM Reply
José Rosales
Antibodies Massive Inmunoproduction Center, IDEA, Venezuela, Venezuela,

Hello, thanks for your question, ok, you can heat the water and place it slowly with the urea, so it will dissolve better and will not change the volume.

7/21/2020 6:18:06 PM Reply

Shammi Akter
Pohang University of Science and Technology (POSTECH)

Hi, Good Afternoon, I have some question about this protocol.

This protocol mention that, After sample load in 18% polyacrylamide gel, run time should keep 45 min under constant 25 A, but according this time sample just inter into the separating gel. So my question is after 45 min, how long I will keep run this gel? My target protein size is 2.5 kDa and 5.6 kDa.

Another question is, At the beginning I started 25 A for 45 min but after gel run some times (around 15 min) later is sowing 16 A and voltage 100. How can I maintain constant 25A? Thank you.

9/24/2020 1:10:31 AM Reply

vu hung
Immanuel Kant Baltic Federal University

I also met problem about voltage, and the answer is u need to change a more power machine electrophoresis and try to reduce your using wells. (5-6 it is enough)

10/20/2020 6:36:50 AM Reply

Shammi Akter
Pohang University of Science and Technology (POSTECH)
Good afternoon, I am trying to make 18 % Acrylamide gel following your protocol. Problem is 30 µl Teamed for 10 ml separating gel is quite high, because of it immediately going to polymerized in the tube. So, can I decreased teamed amount? My another question is, why you used 5 M (3 g) urea? Without urea, there is any problem of protein migration in the gel? My protein concentration is low. Therefore, could I load 50µl protein in the gel?
Thank you.
7/2/2020 10:34:42 PM Reply
José Rosales
Antibodies Massive Inmunoproduction Center, IDEA, Venezuela, Venezuela,

Good morning, you can use less Temed and thus decrease the polymerization time of acrylamity. You can test in a separate tube and time the amount of Temed and polymerization time, before using the crystals. The high concentration of urea helps to define the protein band. Otherwise the band is very diffuse. In effect it can load more volume of your protein and thus increase the possibilities of viewing it. Luck!!!

7/7/2020 7:08:11 PM Reply

Paolo Motta
University of Milan
To whom it may concern,
Good morning, I'm writing because I'm interested in trying your protocol (I need to resolve a 4 kDa protein), but I would like to ask a clarification.
In your Protein Sample Buffer recipe you add 400 mM EDTA, but EDTA solubility in water should be much lower - such a high concentration can be achieved only in very alkaline conditions (3 M NaOH according to Sigma). Is EDTA necessary for a correct run? How could I overcome this problem?
Also, I noticed that you neither add a reducing agent in your Sample Buffer nor you boil your samples, is there a particular reason for this?
Thank you very much for your availability,
Best Regards,

Paolo Motta.
2/25/2019 7:28:13 AM Reply
César Rivera
Energy and Enviroment, IDEA, Venezuela

Good afternoon, we appreciate your interest in our protocol. In the sample buffer the EDTA was used since was the reagent available at the moment, but it can be changed for DTT at the same concentration or β-Mercaptoetanol. EDTA is not necesarry for a correct run, and we did not used reducing agent in the Sample Buffer nor boil the samples since we needed the native proteins. Best Regards.

2/25/2019 11:18:24 AM Reply

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.