Abstract
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
Background
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
Equipment
Procedure
Recipes
Acknowledgments
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.
References
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.
Hi, you're right it's 20 mA of pre-run and then 25 mA of run.
can i ask something else? How did you transfer gel into PVF membrane?? I did electrophoresis quite well but during the process of transferring I lost bunch of proteins How did u detect the protein?
Hi there. I had no problems with the transfer to PVF. The protein was detected with commassie blue. I might suggest that you use nitrocellulose. Ok, when doing the discoloration you have to be careful since the protein is lost. You have to bleach for a short time and watch for your protein to appear. Low molecular weight proteins fade very quickly !! When you see it, you stop fading. In the transfer to PVF, you could use Phenol Red to show that there was transfer. Also if you have an antibody against your protein you could use it.
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.
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,
The pre-run is necessary and very important! Since the gradient is very close to the low molecular weight proteins and when doing the pre-run it moves it away.
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.
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.
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)
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!!!
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.
