Background Information you need to know before start:
1. What is Western Blot?
Please read: Western Blot @ Wikipedia;
you will get every detailed background information from there (history, principle, etc.)
2. What is Western blot used for?
Please search PubMed with key word "western blot", the very first paper on top can give you a general idea of what's the application of Western blot in an experimental setting.
You can also refer to "Molecular cloning: a laboratory manual: Volume 1" if you have further questions.
Before you start: prepare the following equipment and reagents:
- Pipettes (of different volume): to measure reagent
- Reagents that are used for making a gel
- Rack
- Pre-cast cassette (from Invitrogen)
*Please pay special attention to the thickness of the cassette (as labeled out in the picture above). Choose the cassette of proper thickness according to the volume of your samples that need to be loaded.
- And combs
Combs are of different thickness and number of teeth. Depending on how many samples you have and how big volume of each sample you want to load, you should choose different combs accordingly.
Let's get started:
The workflow is as follows
Making lower gel => Lower gel solidification => Making upper gel => Upper gel Solidification
Look pretty easy, uh?
Let me break it down to more details, then you will know where the tricks are:)
Step 1. Make Lower Gel
Lower gel is where protein can be separated by the molecular weight; so depending on the size of your target protein, make lower gel of certein percentage.
Here is the recipe:
The rigidity of gel increase with percentage of gel! (This is important, which we will cover later on.)
Here comes the protein ladder, which can also help you decide what percentage of gel you need to make.
Ok, let's have a small quiz:
If I have protien of molecular weight roughly 31kDa, what percentage of lower gel should I make?
(Answer: 10% or more)
You can see the answer if you select one line below the question:)
Making the gel itself is nothing, you just need to mix everything in that recipe up, then pour the mixture into the cassette.
Two pictures of how to pour a lower gel.
Notes:
1. save the upper 1/4 of the cassette for pouring upper gel (other wise you have no space for protein stacking!)
2. pour the gel immediately after mixing everything up, because TEMED is a super potent catalyzer which will make gel solidify in minutes.
3. gently pour water on top of your lower gel, you can see a boundary between water and gel mixture. Try not to disturbe the boundary. That will be the interface between two layers of gels.
Besides water, you can also pour isopropanol or n-butyl alcohol. It's totally up to what's the tradition of the lab:)) (Trick!Trick!Trick!)
This how lower gel looks like after completion.
Note: once the lower gel solidifies, you will see the boundary between water and lower gel reappear.
Step3: Making Upper Gel
Upper gel is where the volume of your samples get compressed and all the proteins get stacked at the same level, just like a scratch line in 100 meter dash.
Making a upper gel is very much similar to making a lower gel. You just need to mix everything accordingly. No matter what percentage is the lower gel, the percentage of upper gel is always fixed.
Another step that is different from making a lower gel is that you need to aspirate the water out before pour you upper gel. You can use filter paper to suck out the water residue, but REMEMBER NO TO DISTURB THE INTERFACE!
Last but not least: insert the comb into upper gel. Make sure there is no bubble stick to the bottom of comb teeth. (Tricky part: you have to wash the comb and dry it out before use! Dry combs tend to have more bubbles on comb teeth. )
And, eventually, we have this BEAUTY:
Well begun is half done! So start you western blot with a perfect gel:)
