Wednesday, July 31, 2013

Possible mistakes while doing SDS PAGE!

Hi, dear readers, friends, it's been a long time, I wrote here. Sorry for that, was a bit busy in lab! And, you can be happy because of the fact that I was busy, as I got lots of experience to share with you! Let us first start with SDS PAGE!

SDS PAGE - Sodium Dodecyl sulphate - Poly Acrylamide Gel Electrophoresis! This SDS PAGE is done for separating proteins based on their molecular weight. It is a widely used technique and it is very useful for having an idea about the expression of your protein of interest.

Principle:
The name SDS PAGE comes from the fact that this method uses SDS for making your protein uniformly negatively charged and of course, the gel is prepared using poly acrylamide. Why to make the protein negatively charged? Because, here our interest is to separate proteins based only on their molecular weight, but not based on charge and all proteins are not negatively charged like DNA (which is separated based on size using Agarose Gel electrophoresis). SDS is an anionic detergent and it makes your protein negatively charged. Hence your protein moves towards the positive electrode. i.e. anode.

SDS PAGE 

Okay, the principle is fine, but, what are all the possible mistakes that one would do, while doing SDS PAGE? Here, I'll explain some possible mistakes, so that it would help beginners. I recommend you to learn the procedure for SDS PAGE before reading this.

While casting Gel!

  1. The first possible mistake you would do is, regarding the volume of gel that you should prepare! The plates - thick and thin plates. The thick plate comes in different thickness like 1mm, 1.5 mm etc., If you are preparing gel using 1.5mm you need different volume of gel and different volume for 1 mm. So, before preparing the gel, be clear about the volume that you need. If you prepare more, then obviously, its wastage of chemicals, if you prepare less, it is waste of chemicals even now, as your gel won't come up to the top of the plate to dip the comb in (then you must discard and prepare the right volume once again) 
  2. Place the plates right in the gel cast right, check that they are equal i.e not up and down. If you place one slightly up and fix it in the gel cast,  then your gel will leak out! 
  3. Check for leakage always with water before filling your gel in! Don't be over confident that your plates would never leak!
While loading your sample!
  1. Check you are loading your sample only inside the well and not into the tank, because there are possibilities for mis-loading, so double check before releasing your valuable protein sample! (it's valuable isn't it?)
  2. Remember to note down the order in which you load, because there is no point in running gel with out knowing the order of your samples in the gel! This might appear silly, but, happens!
While running!
  1. keep watching your gel, else all your sample would run out! oops, waste of time, chemicals and your hard work!
Wondering? how I'm listing lots of mistakes? Because, I had committed some of these mistakes and I had seen people commiting the rest! Making mistakes, is nothing wrong, but, repeating the same mistake is!

So, remember these things, save your time and chemicals! 

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