SDS-PAGE Basics - Help a n00bie out? (Jun/03/2008 )
Hey everyone.
I've gotten a lot of conflicting information with what I should be doing for my (very first!) SDS-PAGE. I've attempted it twice so far, made mistakes because nobody told me certain details, but I'm hoping the third time will be the charm tomorrow. I haven't been able to see anything on the gel... but I'm doing the gel correctly and running it correctly because the prestained ladder shows up. So that leaves sample prep and staining. All I want to do is roughly determine the purity of the samples, by hopefully only seeing one band. I know there's better methods to do this, but it's more about me learning the technique at this point.
1. What concentration/mass of protein should I be using?
2. How long should I stain with Coomassie? I've heard everywhere from 15 minutes to an hour.
3. I have the protein either in an ammonium sulfate suspension, or just powdered in a jar. It's glucoamylase, if that changes your answer at all. How should I prepare both of those as samples? I dissolved the powdered in MilliQ H2O, and centrifuged the suspension and resuspended the pellet in MilliQ H2O. I was told to try a buffer? What buffer/pH should I go for?
Any other tips/common mistakes would be welcome.
Thanks in advance!
I've gotten a lot of conflicting information with what I should be doing for my (very first!) SDS-PAGE. I've attempted it twice so far, made mistakes because nobody told me certain details, but I'm hoping the third time will be the charm tomorrow. I haven't been able to see anything on the gel... but I'm doing the gel correctly and running it correctly because the prestained ladder shows up. So that leaves sample prep and staining. All I want to do is roughly determine the purity of the samples, by hopefully only seeing one band. I know there's better methods to do this, but it's more about me learning the technique at this point.
1. What concentration/mass of protein should I be using?
2. How long should I stain with Coomassie? I've heard everywhere from 15 minutes to an hour.
3. I have the protein either in an ammonium sulfate suspension, or just powdered in a jar. It's glucoamylase, if that changes your answer at all. How should I prepare both of those as samples? I dissolved the powdered in MilliQ H2O, and centrifuged the suspension and resuspended the pellet in MilliQ H2O. I was told to try a buffer? What buffer/pH should I go for?
Any other tips/common mistakes would be welcome.
Thanks in advance!
It is too late for me to explain in detail, besides, since you are new to the technique, you should afford some reading.
General idea and protocols - http://www.protocol-online.org/prot/Molecu...resis/SDS-PAGE/
Protocols -http://search.vadlo.com/b/q?sn=158621799&a...+page&rel=0
lectures - http://search.vadlo.com/b/q?sn=158621799&a...page+&rel=2
Also check out western blot methods.
2. How long should I stain with Coomassie? I've heard everywhere from 15 minutes to an hour.
3. I have the protein either in an ammonium sulfate suspension, or just powdered in a jar. It's glucoamylase, if that changes your answer at all. How should I prepare both of those as samples? I dissolved the powdered in MilliQ H2O, and centrifuged the suspension and resuspended the pellet in MilliQ H2O. I was told to try a buffer? What buffer/pH should I go for?
Any other tips/common mistakes would be welcome.
Thanks in advance!
1. with coomassie blue, you should be able to see a band with 500 ng. If I were you, I would use more, especially if you want to see contaminations. I don't kow exactly, maybe 1 and 10 µg.
2. If you heat coomassie blue at 50 °C you can stain quickly. I used to warm it few seconds in the microwave (don't boil), and then stain the membrane with shaking, for 30 minutes. It's not necessary to do longer. However I don't like to use heated destain solution to destain quicker, because the destain is not even on all the gel.
Often I destain overnight in a first bath (because it's often the end of the day).
and then I do a second bath of destain the day after.
3. without any information from the provider, I would resuspend in PBS.
apart from the suggestion from the protocols...about your question:
1. Concentration: start by using 1mg/ml (or 1ug/ul) and use also 0.5 and 2 in the same gel...to have a dilution curve
2.Comassie...it depends on the temperature....in general term, if this is your first try, do not worry about overstaining....it could be painful to destain later, but better to have some bands..then you can play..I will suggest at least 1h
3.How much is the NH4SO4? Because it can influence the run..and for the power, check the PI of the protein..and use an HEPES buffer or a Tris-HCl buffer depending on the PI...in the same buffer put also EGTA to chelate divalent ions and that can helps dissolution..because you say that when dissolved in H20 you got a suspension, so I guess is not really dissolved...
hope this can help!
I've gotten a lot of conflicting information with what I should be doing for my (very first!) SDS-PAGE. I've attempted it twice so far, made mistakes because nobody told me certain details, but I'm hoping the third time will be the charm tomorrow. I haven't been able to see anything on the gel... but I'm doing the gel correctly and running it correctly because the prestained ladder shows up. So that leaves sample prep and staining. All I want to do is roughly determine the purity of the samples, by hopefully only seeing one band. I know there's better methods to do this, but it's more about me learning the technique at this point.
1. What concentration/mass of protein should I be using?
2. How long should I stain with Coomassie? I've heard everywhere from 15 minutes to an hour.
3. I have the protein either in an ammonium sulfate suspension, or just powdered in a jar. It's glucoamylase, if that changes your answer at all. How should I prepare both of those as samples? I dissolved the powdered in MilliQ H2O, and centrifuged the suspension and resuspended the pellet in MilliQ H2O. I was told to try a buffer? What buffer/pH should I go for?
Any other tips/common mistakes would be welcome.
Thanks in advance!
It is too late for me to explain in detail, besides, since you are new to the technique, you should afford some reading.
General idea and protocols - http://www.protocol-online.org/prot/Molecu...resis/SDS-PAGE/
Protocols -http://search.vadlo.com/b/q?sn=158621799&a...+page&rel=0
lectures - http://search.vadlo.com/b/q?sn=158621799&a...page+&rel=2
Also check out western blot methods.