I am not entirely sure how to explain what I’ve been doing in the lab without boring you to tears! Prepare to geek out! Instead I’ll post some examples of techniques I’m using to garner some meaningful data. I am currently studying a protein called ‘synaptophysin‘ found in retinal tissue. I’ll leave the hows I go about getting a hold of retinal tissue to your imagination
Basically, I go about a 3 day process from start to finish to complete this task. It involves a couple different assays which is just a fancy term to describe a means to test or quantify a procedure in molecular biology. You can imagine how upset I am if I’ve made a mistake along the way and I get negative data on the 3rd day. In fact, as we speak my holiday plans are being adjusted. Due to an improper transfer from one of my gels to a membrane, I will have to re-run things on Monday and hopefully be able to take a half day on Thanksgiving eve. I started the experiment yesterday in case something didn’t work properly. I ran 2 gels on a Western Blot and one worked great and the other notsomuch.
This is what’s happening at a molecular level and then I’ll explain in a nutshell what I do with it
Ok…so I start with a retina that I harvest from the eye and then drop it into a liquid that I’ve mixed up that causes the cells to lyse or tear apart into little bits. Next I need to sonicate the sample which completely obliterates the cell structures in the retinal tissue. Then I need to rock the sample, use a centrifuge to spin the samples down and pipet off the ‘supernatant.’ I like to think of the supernatant as the “pure stuff” without the tissue pellet or debris from the buffer solution.
Now I have to do some math and I don’t really like that part haha I must run what’s called a protein concentration assay on a 96 well plate like this one:
I load 2 micrograms per microliter of bovine serum albumin in triplicates A through H in the following microliter volumes: .5, 1.0, 1.5, 2.0 etc so if you do that math you get 2micrograms/microliter x .5 (your first loading volume in triplicate) you get 1! Try the next one 2mcg/mcl BSA multipled by 1.0= 2 and so on. This is helpful to determine your place on the microwell plate in case you get lost, you can “retrace yourself” so to speak. You leave A row 4,5,6 empty and this is called “blank.” This is important because you need to have a wavelength reading to make sure the plate isn’t contaminated with anything. Finally, you begin to load your samples once again in triplicates starting in B 4, 5, 6 and so on until you pipet all of your samples onto the plate. The reason behind pipetting in triplicate is so you reduce error and get a better sense of the average protein concentration in that particular sample. You add 2 different reagents to the entire plate with a multiple pipet tool like this one (makes it go sooooooo much faster).
Here comes the math portion. So you need to load into a Western Blot gel 20mcg/mcl of each sample. The plate reader spits out a reading based on whatever wavelength you need it to read measured in nanometers. You use the bovine serum albumin to calculate a standard curve (i.e. a linear value so Y=mx+b remember that from algebra?). You rework the formula to solve for x and you get how many micrograms per microliter of each sample. You simply take 20 and divide it by x (whatever numbers you figure out for each of your unknown samples) and that’s how much protein sample you pipet into your sample prep (this prep has like 3 other steps–more buffer, incubating in a heatblock, vortexing that I will not bore you with).
Now comes the fun part! Pipet a ladder ( a known standard so you can figure out where your proteins are) and then each of your samples into a seperate well on the gel. Typically, I use ones that have 10 wells so depending on how many samples I have I could be running 2-3 gels at once. My protein of interest (synaptophysin) is found at 38kDa or kiloDaltons.
I pour running buffer over the gels and set the power supply to 100Volts for 2 hours! Finally, I take the gel and make a sandwich for the transfer to a membrane paper.
I pour transfer buffer over my casettes and put it in a new box for overnight in a fridge running at 20Volts. This is all in Day 1!!!
DAY 2: First thing, I come in and turn the voltage up to 30volts for 1 hour. Then I must block the membrane in a 5% milk solution that I make from dry milk (you can buy it in a grocery store) and 1x TBST (Tris buffered saline tween-20) that I’ve made. Once again (more math haha) You block the membrane so that the proteins in milk bind to empty spaces where synaptophysin is NOT. Otherwise, when you go to image your membrane it will show up black because the anti-bodies you add to it bind to everything! The blocking takes another full hour, then you rinse it with your TBST solution again. Now it’s time to incubate the membrane for 2 hours with a monoclonal primary antibody. Then more rinsing (3x over 45mins). Finally, you add the secondary antibody and incubate for 1 hour. Then more rinsing (3x over 45mins) Now you are ready to image the membrane using a substrate to develop the membrane! It takes about 3 mins to take the exposure.
I wish I could say it ends there but it doesn’t. In order to quantify your exposures you need to compare where the synaptophysin lies to something else right? The next day I re-probe the same membrane for actin. You’ve heard of actin right? Very important! It’s found at 42kDa. I do the same deal with the 2hr primary antibody, wash x3, secondary antibody for 1hr, wash x 3 and then image. I then use a program called ImageQuant 5.0 to give me values to normalize my data. Synaptophysin divided by actin to give me a number that I average to percent of controls. I get the average for my control group and variable group and calculate the standard error of the mean (standard deviation divided by the square root of n or how many I had in that group). If you made it to the end of this you deserve a star!
Ending notes: 1.I will say this…if I had any idea how much math, molecular biology and chemistry I do day to day I would have never EVER chose this as my major because I would have been too scared! Ignorance is bliss my friends! If I can do it, so can you! It just takes some hard work and determination. You really can do anything you put your mind to doing! Now you have seen what a typical day looks like for me but I really do love my job! It’s so interesting to me and I really take joy in doing these techniques. Maybe, my next work post can be on DNA Fragmentation (another cool thing).
2. Did you know that western blots are used to test for disease? Lyme, HIV etc. Very important translating these techniques into a role in which we as humans benefit!