The overall goal of the following experiment is to assay in vitro the protective effect of the DPS protein by visualizing the reduction in radical mediated DNA damage resulting from DPS activity. This is achieved by first purifying DPS from e coli cells, utilizing several ion exchange columns and ammonium sulfate precipitation As a second step, the purified DPS is added to DNA, which allows for A-D-P-S-D-N-A complex to be formed due to the non-specific DNA binding properties of the protein. Next solutions of Ferris iron and hydrogen peroxide are added to the reaction in order to produce hydroxyl radicals through Fenton chemistry and thereby damage the DNA.
The obtained results show reduction in DNA damage due to the presence of DPS based on the quantification of undamaged DNA bands on agros gels following electrophoresis. Generally, individuals new to this method will struggle with reagent preparation. This institutes unwanted computing reactions such as the formation of ferric iron precipitates, or the spontaneous decay of hydrogen peroxide.
Although this method provides insight into the protective activity of DPS against oxidative stress, it can also be applied to quantify the effects of other proteins and chemicals on DNA degradation. This method can help answer key questions in the field of oxidative chemistry, such as determining the antioxidative properties of cellular extracts for use in the food industry. An essential first step for the DNA protection assay is to obtain DPS protein of high purity.
To begin the procedure for DPS expression and purification, transform a protease deficient strain of e coli with a pet vector into which the DPS protein encoding sequence has been cloned. Inoculate two flasks of one liter LB medium containing appropriate antibiotics with 10 milliliters. Each of an overnight culture grown from a single colony of transformed cells grow at 37 degrees Celsius while shaking to an OD 600 of approximately 0.6.
When cells have reached the desired od induce expression of DPS by adding IPTG to a concentration of 0.3 millimolar incubate at 37 degrees Celsius for three to four hours while shaking harvest cells by centrifugation at 6, 000 Gs for 15 minutes. We suspend the cell pellets in 7.5 milliliters of DEAE buffer, a per liter of induced cell culture at a mixture of protease inhibitors to the pooled cell suspension. To prevent degradation of overexpressed TPS prepare cell-free extract using a French press prime.
The French press with 20 milliliters of DEAE buffer A and then disrupt the sample twice at 20 KPSI centrifuge the lysate at 30, 000 GS for 35 minutes at four degrees Celsius. To clarify insoluble particles save the supernatant, load the cell-free extract into a 30 milliliter DEAE SRO SEAL six B column that has been equilibrated with DEAE buffer a begin to collect the flow through. Once the OD two 80 signal begins to increase above baseline, wash the column with DEAE buffer A until the O od two 80 value returns to baseline while continuously collecting the flow through.
The flow through. Should contain the majority of the DPS protein free from bound DNA, wash the column with 100%buffer B.This eluate contains D-P-S-D-N-A complexes as well as other proteins and can be discarded. The next step is to remove additional contaminating proteins through ammonium sulfate precipitation and is usually carried out in the cold room.
After determining the volume of pooled flow through slowly add dry small grain ammonium sulfate to 62%saturation over a period of 10 to 20 minutes at four degree Celsius while stirring. Well remove precipitated proteins by centrifugation at 20, 000 GS for 30 minutes at four degrees Celsius. Save the supernatant slowly.
Add an additional 227 milligrams of ammonium sulfate per milliliter of supernatant to reach 94%saturation and stir for 20 to 30 minutes. To ensure complete equilibration TPS precipitates at this high salt concentration, collect DPS by centrifugation at 20, 000 GS per 30 minutes at four degrees Celsius. Resuspend the DPS containing pellet in Resus suspension Buffer and adjust the sample volume to 2.5 milliliters.
Remove the ammonium sulfate by buffer exchange using a PD 10 gel filtration column whose gel bed has been equilibrated with 25 milliliters of Resus suspension Buffer. Apply the 2.5 milliliters of DPS sample to the top of the PD 10 column and allow it to soak in, discard the flow through. Collect DPS by eluding with 3.5 milliliters of Resus suspension buffer centrifuge.
The buffer exchanged sample at 16, 000 GS for 10 minutes at four degrees Celsius. To remove any insoluble components, dilute the sample with six to 10 milliliters of dilution buffer to ensure that the salt concentration is low enough for DPS. To bind to the SP SPHEROS column, load the sample onto a 30 milliliter SP spheros fast flow column that has been equilibrated with SP buffer.
A wash with SP buffer A until the OD two 80 trace returns to baseline and discard the flow through. Run a 150 milliliter linear gradient from zero to 100%buffer B while collecting two milliliter fractions. DPS will elute in a sharp peak at around 50%B though variation by 10%is possible.
Check the elucian fractions for DNA contamination by measuring the OD two 60 to OD two 80 ratio. Using a spectrophotometer, the ratio should typically be around 0.7 when the elucian fractions are run on a 15%SDS page gel. The gel should only show one band at around 19 kilodaltons.
Pool the purs DPS fractions after concentrating and exchanging the DPS into a storage buffer. Using a centrifugal filter unit aliquot the purified DPS and freeze in liquid nitrogen for storage at minus 80 degrees Celsius. The genetic protection assay consists of multiple reagents and complex pathogen schemes.
Careful reagent preparation and precise timing are an absolute requirement for reproducibility. The use of a pipetting table is advisable to keep track of steps and volumes during this procedure. Measure 30 milliliters of water into an airtight vial to remove oxygen from solution.
Flush the water with nitrogen for 10 minutes using two syringe needles, one in the fluid and one above it. Add 0.0168 grams of ferrous sulfate heta hydrate to the water to obtain a two millimolar stock solution. Seal the vial and flush with nitrogen for five more minutes.
Then mix. The solution must be clear if any hint of yellow is detectable, prepare a new iron solution. Make a 100 millimolar hydrogen peroxide solution and keep on ice and shield it from light due to spontaneous decay.
Use this hydrogen peroxide solution only for about one to two hours after preparation. Follow an eloqua of purified DPS rapidly in a room temperature water bath and keep on ice centrifuge for 10 seconds at 4, 000 GS in a tabletop pico centrifuge to precipitate aggregates after centrifugation. Measure concentration using a spectrophotometer next dilute linear DNA from a high concentration stock using 12 x reaction buffer to obtain a concentration of 100 nanograms per microliter, pipette enough water into the PCR tubes so that the final reaction volume will be 12 microliters.
Next, create a stock sample of DNA mixed with DPS so that the final doer concentration will be three micromolar pipette the D-N-A-D-P-S mixture into the PCR tubes. Control tubes only receive DNA incubate for 15 minutes at room temperature to allow for DPS to bind to DNA. Add enough two millimolar ferrous sulfate solution to reach the desired concentration quickly add one microliter of the 100 millimolar hydrogen peroxide solution to a 10 millimolar final concentration and incubate room temperature for five minutes.
To allow the Fenton mediated degradation reaction to take place, add one microliter of 20%SDS mix and incubate at 85 degrees Celsius for five minutes to disrupt the D-P-S-D-N-A complexes, the SDS destabilizes, the DPS DNA complex and prevents unwanted gel shifts for the DNA, which improves the ease of quantification. Now incubate on ice for one minute and then add loading dye and load on an agarose gel for electrophoresis stain for DNA using atherium bromide post electrophoresis instead of prior to electrophoresis. This prevents the SDS from interfering with the atherium bromide distribution in the gel.
The intermediate steps of DPS purification analyzed by SDS page are shown here. Lane one is the cell-free extract. Lane two is the flow through of the DEAE SPHEROS column.
Lane three is the EIT of the DEAE column. Lane four is the supernatant of the 60%ammonium sulfate precipitation. Lane five is the DPS sample following 90%ammonium sulfate precipitation and buffer buffer exchange by PD 10 column.
And lane six is the pooled pure DPS fractions. After SP spheros column protein. Molecular weight markers are in lane seven.
The percentages on the bottom of each lane denote DPS purity as determined by image quantification software. The purification process for DPS demonstrated here is very reproducible and protein purity is consistently above 99%No other proteins appear on SDS page gels as visible bands. Purified DPS can be used in a DNA protection assay in which iron catalyzes hydrogen peroxide degradation through Fenton chemistry and produces radical species that directly damage DNA present in the reaction representative results are shown in this figure.
All lanes contain 100 nanograms of linear five kilobase, DNA lane one contains DNA only, and lane two contains DNA with one millimolar hydrogen peroxide lane three contains DNA with one millimolar hydrogen peroxide and 166 micromolar ferrous sulfate. But the DNA has been degraded. Lane's four to eight contain DNA with three micromolar, DPSD demer, one millimolar hydrogen peroxide, and increasing iron concentrations from left to right.
That DPS mediates protection of DNA from oxidative degradation is evident from a comparison between lanes three and five. An overall increase in DNA degradation with increasing iron concentration is also apparent. The results obtained through the DNA protection assay can be quantified through the use of image processing software such as image quant.
This graph shows the result of this quantification averaged over three assays. The error bars indicate the overall high level of reproducibility obtained. Once mastered.
This technique can be performed in about four hours for as many as 40 samples, allowing you to characterize many different conditions in parallel. While performing this procedure, it's important to remember to prepare your reagents accurately and time each step as precisely as possible. Since the assay is quite sensitive to incubation times, After watching this video, you should have a good understanding of how to produce high purity DPS protein and characterize it in an in vitro assay in order to determine its activity in protecting DNA from degradation by reactive oxidant species.
