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Why native gel electrophoresis doesn’t separate effectively by molecular weight

akmay — Fri, 21 Nov 2008 17:54:33 +0000

Native gel electrophoresis does not separate effectively by molecular weight because the native gel electrophoresis technique does not use involve denaturation of the proteins. The proteins retain their folded structure and migrate through the gel according to their mass:charge ratio. The charge of a protein is based on the ionizable side groups of its amino acids. This means that two different proteins with similar molecular weights may travel different distances due to different charges. Using sodium dodecyl sulfate (SDS) in denaturing polyacrylamide gel electrophoresis(PAGE) overcomes this problem because the SDS molecules bind to the protein chains in a constant ratio of about 1.4g of SDS per gram of protein and overcome the charges of the ionizable side groups. This makes the net charge of all the proteins negative so that all the proteins migrate toward the positive anode when the electric current is applied. SDS also changes the native conformation of the proteins into a constant rod-like conformation. This allows the protein chains to move according to their primary structure, and eliminates the differences in shapes that are found in the native secondary and tertiary structures. This allows separation of the proteins based predominantly on molecular mass.

Progress Curves in redox reactions with NADH

akmay — Fri, 21 Nov 2008 17:53:15 +0000

Alcohol dehydrogenase catalyzes the reaction of ethanol and NAD⁺ to form acetaldehyde and NADH. Since NADH is a product, absorbance values go up as the reaction progressed; therefore higher absorbance values mean an increase in activity. Lactose dehydrogenase catalyzes the reaction of pyruvate, NADH and H⁺ to lactate and NAD⁺. NADH is a reactant in this case; therefore lower absorbance values indicate an increase in activity. A progress curve plots absorbance values of NADH over the course of the reaction. The progress curve in an ADH experiment will show an upward trend while the progress curve in an LDH experiment will show a downward trend.

Ammonium Sulfate Precipitation

akmay — Fri, 21 Nov 2008 17:47:27 +0000

A method of protein purification is precipitation an anti-chaotropic salt. Ammonium sulfate can be used to precipitate out certain proteins of interest, such as LDH in protein mixtures. Ammonium sulfate should be added slowly when precipitating a protein because proteins precipitate out at different concentrations of ammonium sulfate. Ammonium sulfate decreases the amount of water available to the protein, causing proteins to precipitate out of solution. Ammonium sulfate is used instead of sodium chloride because it is an anti-chaotropic salt, meaning that it favors protein folding rather than protein denaturation. Sodium chloride is moderately chaotropic and denatures proteins.

Enzyme Activity Assays

akmay — Fri, 21 Nov 2008 16:04:22 +0000

Activity assays are performed to observe how the activity (V₀) varies with different concentration of LDH. The enzyme Lactate Dehydrogenase (LDH) exists in two different forms. The H form is mainly found in cardiac muscle and it is optimized to catalyze the oxidation of lactate to pyruvate. The M form, found in skeletal muscle, is optimized to catalyze the reduction of pyruvate to lactate and the simultaneous oxidation of NADH to NAD⁺. This lab involves purifying the M form of LDH. Because NADH(a reactant) absorbs at 340 nm, measuring absorbance at 340 nm over the course of the reaction will allow us to determine the rate of activity(V₀) for LDH.

Affinity Chromatography

akmay — Fri, 21 Nov 2008 15:59:59 +0000

Blogging is so fun, I can write all kinds of pointless stuff that I enjoy and no one else cares to ever read. So I’ll take some time to share my knowledge of biochemistry, since I know the world is eagerly awaiting my contribution to the internet:

Affinity chromatography is used to purify mixtures of unknown proteins for a single protein of interest. The column matrix contains adenine monophosphate(AMP) immobilized on beaded agarose. The protein sample is diluted before running through the column to bring it down to a measurable range ( < 2 A₂₈₀). When the protein sample is run through the column, lactate dehydrogenase(LDH) binds to the AMP and becomes trapped in the column. The protein sample is followed by elution buffer to “wash the column” and elute out the contaminant proteins that have no affinity for AMP. Next a low concentration solution of nicotinamide adenine dinucelotide(NADH) is run through the column to elute out the proteins that do not bind strongly to the AMP. This “low NADH” is followed by a “high NADH” solution whose NADH concentration is high enough to elute out the LDH. Fraction collector tubes are used to collect a specified volume of solution as it elutes from the column. Measuring absorbance values of these collector tubes at A₂₈₀ allows us to detect the presence of aromatic amino acids, such as phenylalanine, tryptophan, and tyrosine and therefore estimate the amount of protein eluting from the column, following the “washes”.

I hate stupid classes

akmay — Fri, 21 Nov 2008 15:47:44 +0000

I really hate stupid classes. I understand that there’s a need to be well-rounded and whatnot. But, I don’t see why english majors have to struggle through classes like calculus, or why biochemistry majors need to take computer classes. I have no desire to blog about anything, ever. I doubt in my pharmacy career, I’ll ever be quizzed on which html codes to use when posting ordered vs unordered lists. I’m pretty sure all my customers are going to want to know is what to do when their 2-year-old drinks the entire bottle of amoxicillin. And the answer won’t be to go blog about it.

My day

akmay — Mon, 27 Oct 2008 17:15:52 +0000

I have a biochem midterm to study for right now. I also need to take my puppy to the vet. My roommate has mono so I also have to take care of her. I have a million other things I should be doing right now besides this stupid webpage.

Basic Info

akmay — Mon, 27 Oct 2008 17:11:37 +0000

I am a senior at NCSU. I am a biochemistry major. I have 2 siblings. I have a puppy. I live off campus.

Hello world!

akmay — Mon, 27 Oct 2008 06:41:35 +0000

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