SDS-PAGE: Protein Assay

Yesterday (January 31st, 2013), I went to RPI and worked with Eun Ji. But before that, I stopped by the Union Building, got a bit lost, and picked up my ID card (FINALLY!!!! whew...).

When we were heading to the lab, I told Eun Ji that I just did DNA electrophoresis in my bio class. Then, she said that she was just going to do an electrophoresis to confirm her previous data and asked me if I want to do it with her (Of course!!:D)!

Gel electrophoresis is a coomon used technique in biotechnology. It can separate and determine the size of the DNA fragments (cut by restriction enzymes) according to their moving rate. First we load the DNA fragments into a gel well. Then, a positive electric current passes through the other end of the gel. Since DNA fragments are negatively charged, they will slowly migrate to the positive end of the gel at a rate that are proportional to their sizes (the smaller the faster).

However, instead of doing DNA electrophoresis, Eun Ji and I were doing the protein electrophoresis, or SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). Unlike DNA, protein has a very specific 3D shape where all the amino acids are tightly packed. Thus, first of all, we have to make it into a straight chain of polypeptide by adding DTT or other substance, which break the hydrogen bonds between amino acids. Then, we have to add SDS to make the positive-charged protein negative charged because gel electrophoresis works for substance to move from negative end to positive end.

Yet, Eun Ji and I were working on the step before launching electrophoresis - the protein assay, a
simple and accurate procedure for determining concentration of a protein sample. This step is critical because when we are loading the protein into the gel, we want each lane to have the same amount of protein (μg) , not the same volume (μl). Those protein samples are extracted from E. coli. However, while most proteins exist in cytosol, some are in cellar compartments such as inclusion body that are hard to extract proteins. The reason why she wanted to double check her is because in order to get the protein, she has to add SDS to break the inclusion body, which would also affect the targeted protein itself.

Therefore, first of all, Eun Ji got all the materials and the samples she wanted to re-investigate  Also, we want to make sure that we do wear gloves because since SDS affects protein, it can also destroy our skin (It smells really bad, and being exposed too long under it can cause health effects, like I got an headache for just smelling for a few seconds). We divided the 96-well plates into a few sections.

For lane 1,2,3 we added different known concentrations of bovine serum abumin standards as marker (from 0 μg/μl to 5μg/μl). Lane 4, 5, 6 are protein extracted from cytosol, and 7, 8, 9 from inclusion body (which appear blue because of the SDS). Each well in lane 4, 5, 6, 7, 8, 9 were added 10μl of protein samples. Then, we add 200μl of diluted dye reagent to each well, and incubated for about 20 min. The solution turn its color, and we can tell the relative concentration by just comparing the color of the samples to that of the standard. Noted that one can't really distinguish the differences in SDS lanes (7/8/9), so the data analysis wouldn't be reliable.

The next step would be to put the plate into spectrophotometer to measure the absorbance and calculate the concentration. However, because of the shortage of time, I wasn't able to get to this step:( Nonetheless, Eun Ji explained the following steps, converting volume, loading and running gel, and analyzing gel, in which the whole process may take up to 2 days!:O To get the specific target protein even involves more steps!

Later in the day Eun Ji sent me the data including some comments. In general, I did fine on it, but compared to hers, my numbers fluctuate a lot. One thing I would need to work on is the pipette technique, especially when dealing with such tiny amount of substance. Meanwhile, I asked Eun Ji why SDS-PAGE runs vertically where as DNA eletrophoresis runs horizontally. She was kind of surprise because she has never thought about this before!! In the email, she explained that SDS-PAGE is consist of two layers (upper layer (stacking gel) and lower layer (separating or running gel), and vertical direction just makes the process easier. Another reason that I found online is that since the gel runs best in anoxic environment, it's better to have the protein in sealed b/w 2 layers rather than open layer like that in horizontal one.

Anyways, I really learned a lot of lab techniques yesterday, and Eun Ji was always really patient and did a good job explaining things to me. Next week I can finally get into CBIS on my own!!!:D