Question

2. Protein purification
a) Determine the subunit composition of a protein from the following information:

Molecular Mass by Gel Filtration: 200 kD

Molecular Mass by SDS-PAGE: 100kD
Mass by SDS-PAGE with 2-mercaptoethanol

   40kD and 60 kD

b) In what order would E, H, and V be eluted from a DEAE column at pH 8?

c) You have a mixture of 3 proteins, one is 100 amino acids long, another 350 amino acids long, and the third is 600 amino acids long. Sketch the chromatogram ( a plot of elution time/volume as a function of A280) for a gel filtration chromatography experiment to separate these three proteins. Assume each protein is equally abundant and has a statistically normal number of tryptophans.

d) what would you do to validate the chromatographic observations in c) above?

*Please answer b thru d*

Answer 1

So here's a detailed explanation of your question

a)

The protein contains 2 60 kD polypeptides and 2 40 kD polypeptides. Each 40-kD is disulfide-bonded to a 60kD chain The 100 kD units associate non-covalently to form a protein with a mass of 200kD

Let us discuss that step by step:

1. the apparent false positive of the Bradford assay; does not mean you have proteins because also the buffer components react with bradford reagents. to exclude that you need to check if the elution buffer give the same color with bradford as the samples.

2. on your SDS-PAGE there were no bands in the wash and elution fractions, while there were bands in the flow-through (including that of my protein). that could means only one thing there was no binding with IMAC and I suggest that you check your protocol or change the methods, in the past I had great experience with Ni-NTA agarose beads if you are purifying a his-tagged proteins.

3. in case if you are sure about the IMAC protocol and that binding with your protein, then the problem is the protein concentration and sensitivity of Coomassie blue, I would suggest to use stain-free gel as the sensitivity is much higher than coomassie and also does not require any staining/destaining time.

Purification by SEC To perform a separation, the medium is packed into a column to form a packed bed. SEC media consist of a porous matrix of spherical particles with chemical and physical stability and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer, which fills the pores of the matrix and the space between the particles. The liquid inside the pores, or stationary phase, is in equilibrium with the liquid outside the particles, or mobile phase. Samples are eluted isocratically so there is no need to use different buffers during the separation. However, a wash step using the running buffer is usually included at the end of a separation to remove molecules that might have been retained on the column and to prepare the column for a new run. SEC can be used directly after IEX, CF, HIC, or AC since the buffer composition will not generally affect the final separation.

Group separation SEC is used in group separation mode to remove small molecules from a group of larger molecules and as a fast, simple solution for buffer exchange. Small molecules such as excess salt or free labels are easily separated from larger molecules. Samples can be prepared for storage or for other chromatography techniques and assays. SEC in group separation mode is often used in protein purification schemes for desalting and buffer exchange. Sephadex G-10, G-25, and G-50 are used for group separations. Large sample volumes, up to 30% of the total column volume (packed bed), can be applied at high flow rates using broad, short columns.

High-resolution fractionation SEC is used in fractionation mode to separate multiple components in a sample on the basis of differences in their size. The goal can be to isolate one or more of the components, or to analyze the molecular-weight distribution in the sample. Optimal results for high-resolution fractionation will be achieved with samples that originally contain few components or with samples that have been partially purified by other chromatography techniques to eliminate most of the unwanted proteins of similar size. High-resolution fractionation by SEC is well-suited for the final polishing step in a purification scheme. Monomers are easily separated from aggregates. Samples can be transferred to a suitable buffer for assay or storage. Prepacked SEC columns are highly recommended for optimal performance during high-resolution fractionation and are available for three different purposes: • Preparative purification for sample volumes in the milliliter range. • Small-scale preparative purification for sample volumes up to 500 µl. • Analytical runs for sample volumes up to 500 µl. HiLoad™ columns prepacked with Superdex™ prep grade media, or HiPrep™ columns prepacked with Sephacryl™ media, are used for preparative purification, which is characterized by collection of the sample. The sample volume is in the milliliter range and the collected fractions are usually in milligram amounts

Rapid purity check and screening Short columns with small bed volumes such as Superdex 75 5/150 GL, Superdex 200 Increase 5/150 GL, and Superose 6 Increase 5/150 GL are suitable for rapid purity check or size analysis of proteins and other biomolecules. Short cycle times, together with small sample volume and low buffer consumption make this column a good choice in screening experiments to check protein homogeneity. However, when using the same media, shorter columns give lower resolution than longer columns. Resolution in SEC The success of SEC depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation. After selection of SEC medium, sample volume and column dimensions are the two most critical parameters that will affect the resolution of the separation. Chromatography system-related factors can also affect resolution

Sample volume and column dimensions The sample volume can be expressed as a percentage of the total column volume (packed bed). Smaller sample volumes help to avoid overlap if closely spaced peaks are eluted. Figure 1.3 illustrates how sample volume can influence a high-resolution fractionation. For group separations, use sample volumes up to 30% of the total column volume. For high-resolution fractionation, a sample volume from 0.5% to 4% of the total column volume is recommended, depending on the type of medium used. For most applications the sample volume should not exceed 2% to achieve maximum resolution. Depending on the nature of the specific sample, it might be possible to load larger sample volumes, particularly if the peaks of interest are well resolved. This can only be determined by experimentation.

let me know if I can help you with anything.

best of luck,