Dear Mirreille

differently to what do you think is it not completelly true that agarose gel is for dna and poliacylammide gel are for proteins.

In some cases poliacrilammide gels (native) can be also used for dna (small fragments)

https://eu.idtdna.com/pages/education/decoded/article/running-agarose-and-polyacrylamide-gels

However the main difference beetween the agarose and acrylammide regards the pore size. Agarose permit the formation of bigger pores and can be used to solve bigger molecule as dna while acrylammide has smaller pores and it is able to solve small molecule as dna fragments or proteins.

just to make you clear:

1 aminoacid has a molecular weight of about 100Da

1base of dna has mw of about 650 Da

and in case of double strand dna the size is double

and while proteins >1000aa are quite rare, for dna size >1000Da are quite common (eg a standard plasmid for cloning and expression in e.coli is about 5000Da)

therefore two molecules with so different size need gels with different resolution.

Moreover while dna is intrinsically negativelly charged this is not true for proteins that with-out sds (native gel) do not run in function of their mw.

if you are interested you can find some more conformation about sds page on my blog: https://proteocool.blogspot.com/?m=1

ProteoCool n 15: Overview on sds-page at page 3

up to know i did not write nothing regarding agarose gel. the only advice that i would like to provide you is

DO NOT USE ethidium bromide. it is very toxic. there are Now many alternatives eg Gel Red (biotium) Gel Star (Lonza) that work well and are less dangerous.

agarose gel is generally cheaper, simple to be prepared and you can stop it, give a look and restart while the acrylammide once is stained cannot be re-started.

sorry for the long answer.

good luck

ciao

Manuele

Dear Mirreille

differently to what do you think is it not completelly true that agarose gel is for dna and poliacylammide gel are for proteins.

In some cases poliacrilammide gels (native) can be also used for dna (small fragments)

https://eu.idtdna.com/pages/education/decoded/article/running-agarose-and-polyacrylamide-gels

However the main difference beetween the agarose and acrylammide regards the pore size. Agarose permit the formation of bigger pores and can be used to solve bigger molecule as dna while acrylammide has smaller pores and it is able to solve small molecule as dna fragments or proteins.

just to make you clear:

1 aminoacid has a molecular weight of about 100Da

1base of dna has mw of about 650 Da

and in case of double strand dna the size is double

and while proteins >1000aa are quite rare, for dna size >1000Da are quite common (eg a standard plasmid for cloning and expression in e.coli is about 5000Da)

therefore two molecules with so different size need gels with different resolution.

Moreover while dna is intrinsically negativelly charged this is not true for proteins that with-out sds (native gel) do not run in function of their mw.

if you are interested you can find some more conformation about sds page on my blog: https://proteocool.blogspot.com/?m=1

ProteoCool n 15: Overview on sds-page at page 3

up to know i did not write nothing regarding agarose gel. the only advice that i would like to provide you is

DO NOT USE ethidium bromide. it is very toxic. there are Now many alternatives eg Gel Red (biotium) Gel Star (Lonza) that work well and are less dangerous.

agarose gel is generally cheaper, simple to be prepared and you can stop it, give a look and restart while the acrylammide once is stained cannot be re-started.

sorry for the long answer.

good luck

ciao

Manuele

Manuele Martinelli thank you very much for this very explicit answer and for all your advice (i am shocked now) ; as the perfect beginner i usually use Ethidium bromide for my gel. Thank you for giving me others alternatives …...

I will read your blog for more informations.

Dear Mireille

i don't won't scare you.

I used ethidium bromide for many years and i'm still good.

But it is a cancerogenic compounds that need to be manipulate with attention especially if you are preparing solutions by yourself and now that are available alternatives less toxic that are not so expensive is it better to leave it for the others.

but don't be scared from my reply!!

Manuele

Because the range of pore sizes agarose offers is less convenient for separating most monomeric proteins than those offered by polyacrylamide. Also, because you can include SDS with polyacrylamide, thus enabling the electrophoretic separation of proteins on the basis of molecular weight alone. As far as I know this is not possible or at least common with agarose.

Regarding ethidium bromide, make sure you stick to local safety regulations but don't be shocked/scared; there's no reason to. I have never seen primary data proving that EtBr is a carcinogen, although of course I'd love to see some if only to vindicate the safety measures I have been following for years.

Hope it helps!

Alejandro Martin and Manuele Martinelli your words helps me considerably to understand the different use of these gels. Thank you for sharing your experiences. I will be more consciencious while handling EtBr from now.

You may use agarose or starch for gel electrophoresis of proteins. You may have to vary the concentration of agarose to determine the pore size that you need for your protein of interest.

Thank you for your answer Phillip Esbenshade

Mireille Kameni

The first step in running a denaturing gel is to denature your proteins. This is accomplished using: SDS ,SDS is the main star of the denaturing protein gel. SDS is a detergent composed of a hydrophobic hydrocarbon tail attached to an ionic sulphate group and a key component of loading buffer,. When SDS meets up with your protein, SDS’s hydrocarbon tail dissolves any hydrophobic region of the protein, while the sulfate end breaks non-covalent ionic bonds. This causes your protein to lose its secondary and tertiary structure, and well…unfold. Once surrounded by SDS, your previously carefully folded protein becomes loose and long, just like overcooked spaghetti. Following this will be an addition of a reducing agent and heat. These are furtherly helps to completely denature the proteins and also helps with physically loading the gel. Now that your proteins are nice and linear, it’s time to run them out on your polyacrylamide gel using electricity. That's why we now use gel eletrophoresis to actually view the proteins fragments or bands. Remember, your proteins won't move through the gel by their own volition; instead you must subject your gel to an electric current, with the negative charge at the top where the proteins are loaded and the positive charge at the bottom. SDS-coated proteins have a large negative charge (thanks to the SDS), thus the proteins are attracted to the positive charge and move from top to bottom. I hope this answers your question.

DNA based on charge, molecules are separate, the movement is also negative to positive side according to electrophoresis principles. But in case of protein it is not possible because both charges present in molecules. So first through anionic detergent SDS you will make the net protein charge as negative then run the electrophoresis and the separation will occurs properly based on charge and size.

The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis. However, the migration of DNA molecules in solution, in the absence of a gel matrix, is independent of molecular weight during electrophoresis.To separate molecules based on their lengths, samples are run in denaturing conditions. For proteins, sodium dodecyl sulfate (SDS) is used to linearize proteins and to negatively charge the proteins. ... Protein Ladder is the must in SDS-PAGEto determine the molecule weight of the protein of interest.

thank you for your answers Dear Audrey Vanya Sailaja Inampudi Eman Bassiony

Mostly concerned with size of the molecule. DNA is a high molecular weight molecule.It need pore size should be high compare to PAGE.

One more answer for your question here. For protein in SDS gel, we normally run longer time right. If you use agarose gel, it will melt before your getting your results. Sometimes, at the end of SDS page running time, you may notice the running solution is hot.

Ming Chuan Yu effectively i noticed that! thank you for your contribution

Amongst others, SDS PAGE is nessesaary for protein analyses to create a charged setting to analyse protein features (Mol whight, size, phosphorylation sate ...) on a reliable and uniform basis.

Agarose is used for comparable reasons. Morover, the differences in size are important (comparing proportions of proteins vs DNA).

I must sadly declare that Electrophoresis is not reliable method at all, and not the convenient method.

Then, reliable and quantitative HPLC-photometric method is recommended in Clinical and Biochemical studies (please see file; SEC column 300A silica).

Protein microsequencing, if protein is covalently bound, is also quantitative and reliable method (please see files; HepG2 fucoidan and JMBT alopecia).