hi I'm Steve freebie I am a staff scientist at biorad labs in this video I am going to demonstrate how to run good reproducible 2D gels the video is going to be broken into three segments the first segment will be how to apply sample and rehydrate the ipg strip the second segment will be on how to equilibrate the ipg strip trips in preparation for running on the second dimension gel and the third segment will be staining of the second dimension gel in this video I hope that you get tips and techniques and tricks that will is help you run good quality two Dimension gels after sample preparation the first step to 2D is to rehydrate the ipg strip and I'm going to demonstrate this process using bior Rad's 2D starter kit the 2D starter kit is a great tool for learning 2D gel electropheresis it contains all the reagents and an ecoli sample for running a 2d gel the first step is to prepare the sample and the reagents according to the instructions in the manual the components of the rehydration buffer are Ura chaps DT ampol and bromophenol blue I will be loading 200 microgr of eoli sample that has been reconstituted in rehydration buffer onto an 11 cm ph4 to7 ipg strip to rehydrate the strip I will load 200 microl of sample into the focusing tray in each lane that I'll be running a strep I am using a focusing tray that is specific for 11 cm streps trays are available for all ipg strip lengths tilt the tray toward you to allow for even distribution of the strip an easy way to do this is to take the focusing tray put it on the edge of the lid to the focusing tray and you have the tray at an angle toward you dispense sample along one edge of the lane of the tray and you want to dispense it in a nice even bead from one electrode across to the other one even bead I have shown you this with the focusing tray but this can also be done with the rehydration tray now we are ready to place the IP strip into the focusing tray an IP strip has two sides one is a plastic protective backing and the other is a dried gel You Begin by removing the plastic protective backing with forceps and you can tell where that plastic protective backing is by a slight indent on one side of the plastic pieces the side that has indented is your protective backing you pull that rapidly across and now you have gel Side Up take your forceps hold onto the strip on the negative side you'll see the other side has a positive side the negative side is opposite of that and you can always tell when the ipg strip is facing down be build to read the numbers of the PH range of the ipg strip in the correct orientation now the gel will be loaded onto the sample bead gel side down if you are running multiple samples I recommend labeling your strip before the step make sure to label the side of the gel and not the protective backing to load the ipg strip into the focusing tray place the positive end of the the ipg strip into the positive electrode of the focusing tray and slowly drop your ipg strip into the lane of the focusing tray being very careful not to trap any air bubbles if there are any air bubbles trapped you can pick up the end and gently reposition the ipg strip until the air bubbles are expelled if you don't remove air bubbles there will be areas in the ipg strip that do not not rehydrate properly and that will cause serious anomalies in your First Dimension isolet focusing run the initial step of the First Dimension separation is to rehydrate the strep I will do passive rehydration in the focusing tray other forms of rehydration are active which may be used for high molecular weight proteins and cup loading which is ideal for the separation of proteins that will be separated at extreme basic and acidic pH place the focusing tray into the protein if Cell making sure to align the positive electrod with the focusing tray with the Positive electrode of the protean if cell and add enough mineral oil to cover the strip in each lane the purpose of the mineral oil is to prevent the strips from drying out during the course of the rehydration and the run now I am going to program the protein if Cell instructions for programming are provided in the protein if Cell manual typical rehydration times are 12 hours and focusing time ranges from 6 to 8 hours at the completion of focusing if you do not have time to run the second dimension gel first remove the strips from the focusing tray blot off the excess mineral oil and place the strips into a rehydration tray at minus 80° C the steps following rehydration and focusing of the ipg strip our equilibration of the ipg strip in preparation for the second dimension gel which I will now demonstrate the equilibration process is a two-step procedure equilibration buffer number one contains dtt for reduction of proteins in the strip equilibration buffer number two contains iodo camide for alkal of proteins in the strep each solution contains SDS for denaturation of proteins in the ipg strip to ensure Optimal Performance in the second dimension prior to equilibration I will remove the ipg strip from the focusing tray and blot the excess oil off of the strip first pick up one end of the ipg strip turn it over so the gel side is up and place the ipg strip gels side up on a dry piece of filter paper wet another piece of filter paper and place the wet filter paper onto the ipg strip and gently blot the excess mineral oil off the strip be careful not to press too hard to damage the ipg strip gel the next step is to equilibrate the streps I prepared buffers from the ready prep 2D starter kit as instructed in the manual remove the wet filter paper from the top of the ipg strip place the ipg strip into a lane gels Side Up of the rehydration tray add 2 ml of equilibration buffer number one to each lane of the rehydration tray that you have a strip place the rehydration tray onto a Shaker roer table for 15 minutes note that the rehydration tray has an angled end and a straight end whenever you're pouring out liquids use the Straight end so that it does not slide out of the tray now add 2 ml of buffer number two shake for 15 minutes and pour out at this point the strips are ready to run on the second dimension gel I am going to use bior Rad's Criterion pre-cast gels which accommodate the 11 cm ipg strips that we ran earlier prepare the gel By REM moving the ceiling tape from the bottom of the gel and slide out the comb from the top rinse the wells with good quality di water tap the water out into a beaker and then place the gel sideways and remove with the piece of filter paper the excess water from the wells be careful not to touch the bottom of the gel as it can damage the surface of the second dimension gel we usually do this because unpolymerized acrylamide can cause anomalies in your second dimension gel if it's not removed to remove the strip from the tray place the forceps on the tray and gently push on the plastic backing of the gel to push the strip towards the angled Edge lift up the strip from the right tab I am going to rinse the strip off and running buffer that I have previously prepared dip two or three times this will remove a cooler bration buffer number one and two that may still be on the surface surface of the ipg strip place the ipg strip gel Side Up place the plastic backing against the back of the cassette position the strip centered in the middle of the well with your forceps and gently push on the Strip until it touches the gel and you can tell when it's touching because you'll feel a a little bit of springiness a little bit of resistance don't push too hard now I will insert a Precision plus standard plug which is a broad rrange standard that has been embedded in auros into the reference well on the right side of the gel break off one plug hold on to the handle and break off the spatula and using the spatula push out the auros plug for Criterion gels I usually cut the plug into four slices to match the size of the well in this case I need one with forceps take one of the slices place on the back of the gel cassette over the reference well and gently push the plug into the reference well at this point we want to seal the ipg strip and standard gel plug into The Well of the gel I will use prodium plus overlay auros heat in the microwave oven until it boils and when the temperature of the auros is no longer hot to the touch take the hot auros and fill up the well and do this in one rapid action side to side working quickly use your forceps to push the ipg strip against the gel to push out any air bubbles auros is going to solidify fairly fast you can see the auros filling in the place of the Bubbles as they get pushed to the top small air bubbles are of not an issue try to get some of your larger ones and you should not have any problems for your second dimension gel now that the auros is solidified I'm ready to run the gel in this case I'm going to run the gel in the Criterion gel box which holds two gels in most cases you're going to run multiple gels and you want to use a criterian DOA cell remove the gel cassette and place it into the Criterion cell cell load the upper buffer chamber with running buffer place the lid onto the Criterion cell insert the leads into your power pack power supply program for 200 Volts for 1 hour constant voltage and start the second dimension gel has now completed running you know it is finished when the die front has reached the bottom of the gel we're now going to remove the gel from the Criterion cassette and prepare the gel for staining remove the gel from the gel box pour out the upper buffer and open the gel by using forceps or a similar tool by pushing the forceps against the plastic cassette towards you at the top and work your way down so fold open like so after I open the gel cassette I use forceps to remove the ipg strip from the surface of the gel starting at the bottom of the gel gently pull the gel away from the surface of the cassette the easiest way to do this turn the gel upside down over the surface of the staining solution remove a corner and remove a corner until the gel falls into the solution biorad offers many different staining choices for 2D gels ranging from the visual stains the traditional kamasis and silver stains to the fluorescent stains Flamingo and cypro in this case I am staining this gel with biosafe Kumasi and the first step requires three 10minute rinses and water to remove SDS from the gel we are now going to stain the gel using bior Rad's biosafe Kumasi stain following the three 10minute water washes we're going to transfer the gel to a tray of biosafe Kumasi and we are going to stain the gel for 45 minutes to 1 hour following staining we are going to transfer the gel from biosafe kamasi back into water and we are going to Dain until you get desired background at that point the gel will be ready for Imaging here you can see that I have generated a gel with a clear background and well-resolved spots I have now completed the entire process for running a 2d gel I hope that the techniques and tips will help you when you run your next 2D gel as you have seen clean sample and the biorad 2D stutter kit will help you generate good quality reproducible 2D gels for additional information and techniques and tips on Sample preparation and 2D gel electris you can visit our expression proteomics website at www expression proteomics