GRACE+T

**Title**: Specific Protein Identification Techniques **Introduction: ** Purifying, detecting, and characterizing proteins Detecting can be considered part of characterizing. Expression of the protein is also important  are important procedures that must be accomplished before the structure and mechanism of a protein’s actions can be studied [1]. In order to produce recombinant protein, many decisions what decisions?  must be made upon the procedures that would provide optimal conditions for protein growth because proteins vary in size, charge, and water solubility [1]. Expression in E.coli is most common and suitable for bacterial growth "suitable for bacterial growth" doesn't make sense. Why is expression in E. coli most common?  [2]. In the purification procedures, it is ideal to have strong, specific binding, and mild elution conditions there are many purification techniques, which one are you talking about?  [2]. The type of resin and buffer used is also significant [2] why is it significant? . Technology is also used, and variations in the calibration, model, or brand can alter results This sentence is way too general . These decisions What decisions?  must be considered, and options what options?  examined before one decides how to prepare the target protein for protein assay. The objective of the lab is to express transform  the plasmid into the BL21 (DE3) bacterial, purify it using column chromatography, and characterize don't forget about expressing it  it to produce a pure pGEM-gbr22 pGEM is a plasmid, not a protein  protein sample. It is hypothesized that the end result would produce a sample that is purely the target protein. If done correctly, the gel should show one protein band within the sample analyzed. **Materials and Methods: ** <span style="font-family: Cambria,serif; font-size: 11pt;">All bacteria and solutions should be put on ice don't need to mention this <span style="font-family: Cambria,serif; font-size: 11pt;">. 25 uL of E. coli BL21 (DE3) bacterial cells were added to each of 2 transformation tubes. pGEM-gbr22 plasmid DNA were spun down with a mini-centrifuge. 1-2 uL were added into the tubes. Amounts should be listed as concentration, not as absolute amounts. That way other scientists can scale up your experiment with ease. <span style="font-family: Cambria,serif; font-size: 11pt;"> Tubes were iced for 30 minutes, and 2 plates of agar with Ampicillin antibiotics were incubated. Then, the tubes were heat shocked in a 42 oC water bath for 45 seconds, and transferred on ice for 2 minutes. 200 uL SOC media was added. Tubes were in incubator for 30 minutes at 37 oC at 250 rpm. 50 uL of each tube was pipetted onto each plate evenly. Store in 37 oC incubator overnight. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">10uL of ampicillin was added to two sterile 14mL tubes of 5mL LB. A colony of bacteria was added to each LB tube. Both were capped and placed in the shaking incubator at 37 oC and 250-350 rpm for about 8 hours. Culture plates were wrapped and stored in 4 oC fridge. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">25 mL of the fresh LB, 50 uL of ampillicin, and 0.625 mL of the culture were transferred to a sterile 125 mL flask, covered with foil, and secured into the shaking incubator, set at 37 oC and 200-350 rpm for 16-24 hours. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">The bacteria was poured into a 50mL conical tube and centrifuged at 5,000 rpm for 10 minutes at 4 oC. Liquid was decanted and the pellet was weighed. 2.5 mL of 1x PBS was added to the cell pellet and vortexed. 51 uL of lysozyme was added and vortexed. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">A wash buffer of 10mL 1x PBS and 20mM of 1M Imidazole, and a 10mL elution buffer of 1x PBS and 250mM of Imidazole were prepared. 1uL of cyanase was added to the culture and mixed. The lysate was centrifuged at 4 oC and 14,000 rpm for 20 minutes in a 1.7 mL tube. The lysate was filtered with a 0.45 um SFCA syringe filter with a 5mL syringe into a 14mL round bottom tube. 0.5 mL of Ni-NTA resin/buffer mix was added to the supernatant. The Bio-Rad Econo chromatography column was used and ran through with the wash buffer, then the Elution buffer twice. The Nanopure system was used to find the concentration of the Elution samples. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">The samples were loaded into the gel wells, and ran for 25 minutes at 200 V. The gel was rinsed multiple times and stained using an Imperial protein stain for 1.5 hours. It was then rinsed over night. Then it was dried at 75 oC on Gradient cycle for 1.5 hours to produce the final gel. Your M&M is a little too specific, you don't need to include things such as "both were capped and placed in the shaking incubator", or "the pellet was weighed". Don't forget to include company and product info. **<span style="font-family: Cambria,serif; font-size: 11pt;">Results & Discussion: **

<span style="font-family: Cambria,serif; font-size: 8pt;">Figure 1: Agar growth plate of LB media with Ampicillin. Lid is off, so no label is shown. Bacteria is E. coli BL21 (DE3) and there is no plasmid (No DNA control). Culture stored on 3/5, picture taken 3/6. Don't need to include dates

<span style="font-family: Cambria,serif; font-size: 11pt;">No colonies grew.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: justify;">Figure 2: Agar plate with LB media and Ampicillin. Lid is off, so no label is shown. Bacteria is E. coli BL21 (DE3) and plasmid is pGEM-gbr22. Culture stored on 3/5, picture taken on 3/6. <span style="font-family: Cambria,serif; font-size: 11pt;">There were around 50 colonies of bacterial, with unexpressed pGEM-gbr22.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: justify;">Figure 3: Agar plate with LB media and Ampicillin. Bacteria is E.coli BL21 (DE3) and plasmid is pGEM-gbr22. Belonged to another group. Culture stored on 3/5, picture taken on 3/6 <span style="font-family: Cambria,serif; font-size: 11pt;">This plate had sufficient amount of colonies that were purple, indicating that they had enough time for the pGEM-gbr22 plasmid to be expressed.

<span style="font-family: Cambria,serif; font-size: 8pt;">Figure 4: 125 mL Erlenmeyer flask with 25 mL of fresh LB solution, 50 microliters of Ampicillin, and 0.625 mL of starter culture. Culture contains E.coli BL21 (DE3) bacteria and pGEM-gbr22 plasmid. Picture taken on 3/7 mention the purpleness



<span style="font-family: Cambria,serif; font-size: 8pt;">Figure 5: Wet protein pellet. Weight: 0.89 g. Pellet contains E. coli BL21(DE3) bacteria with pGEM-gbr22 DNA after being centrifuged. LB and Amp media was decanted before being weighed, Picture taken on 3/7. mention the purpleness

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 6: Elution 1 in a 10mL round bottom tube, containing pGEM-gbr22 protein in an Elution buffer.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 7: Elution 2 in a 10mL round bottom tube, containing pGEM-gbr22 protein after a second wash in the Elution buffer.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 8: The absorption spectra at 280 wavelength setting from Nanodrop; the first of 2 trials.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 9: Absorption spectra in the UV/VIS setting from Nanodrop. Readings were at 574 nm and 280 nm; the first of 2 trials.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 10: Gel after overnight wash and before drying; 6 samples were in the wells, along with a Bio Rad Protein Ladder [PageRuler Prestained, 26616, BioRad] for reference. Label your lanes like this: Lane 1: skipLane 2: ladderLane 3: lysateetc...

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 11: SDS-PAGE band profile of the Thermo Scientific PageRuler Prestained Protein Ladder for the reference band (#26616). Image from a 4-20% Tris-glycine gel and subsequent transfer to membrane. Image from website.

<span style="display: block; font-family: Cambria,serif; font-size: 8pt; text-align: center;">Figure 12: Gel after drying at 75 oC for 1.5 hours. The sample number and reference Bio-Rad Protein Ladder is labeled below. <span style="font-family: Cambria,serif; font-size: 11pt;">The lysozyme used in purification will digest the protein cell walls. The cyanase is a nuclease that will break down the nucleic acids of the cell. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">Each pGEM-gbr22 is modified to be connected to a HIS tag, which identifies it as the target protein. When Ni-NTA resin/buffer mix was added, the HIS attached strongly to the nickel, which is connected to a large bead. By adding Imidazole to the solution, the Imidazole competes with the HIS for nickel bonding, allowing for the release of the pGEM-gbr22 protein. <span style="font-family: Cambria,serif; font-size: 11pt;">The wash and elution buffers differ in concentration. The wash buffer has a 20mM of Imidazole, while the elution buffer has a 250mM concentration of Imidazole. When the wash buffer was added to the protein and Ni-NTA resin/buffer mix, only a small amount of Imidazole was available to compete with the HIS tag. When the elution buffer was used, there were more Imidazole, causing the HIS to release the Ni and fall through the column, collecting in the Elution 1 and 2 samples. What is the significance of the low concentration imidazole? <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">From the purification lab and Nanodrop readings, bacterial yields were collected using Beer’s Law (A=Ebc). <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">Yield at 280nm wavelength, using a set extinction coefficient and absorbance values from the Nanodrop: C= (0.253+0.13)/(38,850*1) = 4.93E-6 M * 25,794.2 (g/mol) = 0.12717 (mg/mL) *5mL = 0.63585 mg

<span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">Yield at maximum wavelength: <span style="display: block; font-family: Arial,sans-serif; line-height: 24px; text-align: justify;">C= (0.26+0.27)/ (118,300*1) = 2.28E-6M * 25,794 (g/mol) = 0.0589 (mg/mL)*5mL= 0.2956 mg

<span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">Samples were taken throughout the lab. Sample 1 contains all the proteins grown from the original culture. Sample 2 contains the supernatant after centrifuging, carrying cell debris. Sample 3 contains the waste liquid, filled with proteins smaller than the pGEM-gbr22 that did not bind to the Ni-NTA resin/buffer mix. Sample 4 was collected after the wash buffer, allowing a small amount of the target protein to flow down. Sample 5 and 6 were collected after each elution buffer, and should contain only the target protein inside. This should be in your captions. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">From the gel, using the BioRad Protein Ladder as reference, it is determined from reading the protein bands of Sample 5 that the protein is at 25 kDa. The protein’s true weight is 25. 794kDa. This is fairly accurate. However the sample was not as pure as ideal. There were 4 total protein bands in the gel reading, giving 25% purity. This implies that there were contaminations. This could be due mixing of the gel’s wells. Also, if an insufficient amount of pGEM-gbr22 proteins were collected, it would not show up as well on our gel. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">Other errors would that could have caused contamination includes non-sterile technique, like not turning on the flame beside the experiment to rid of outside bacteria. Time constraints were also an issue, as seen with the bacteria culture that was not grown long enough to see purple protein expression. Extra attention should also be place upon durations of heat shock and times outside of the ice bucket, which would both cause the protein to denature. Potential errors can also be variations in buffer and stock concentration, as well as variations in technology.

You can also include analysis on the other lanes of the gel. **<span style="font-family: Cambria,serif; font-size: 11pt;">Conclusion: **

<span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">E.coli BL21 (DE3) bacterial cells were expressed with pGEM-gbr22 plasmid DNA should read: "GBR22 protein was expressed in E. coli (BL21) with pGEM-gbr22 plasmid DNA" <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">, purified and isolated using column chromatography, and characterized using gel electrophoresis, allowing for the prediction of their molecular weight and the purity of the sample. The sample from the gel had contamination and was less pure than ideal and the protein weighs about 25 kDa. After protein characterization, a protein assay fast protein liquid chromatography, not an assay. Assays are used to check for activity. <span style="display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;"> would be necessary in order to isolate sufficient amounts of a given protein to be used in later studies [1]. **<span style="font-family: Cambria,serif; font-size: 11pt;">Reference: ** <span style="font-family: Cambria,serif; font-size: 11pt;">[1] <span style="color: #222222; font-family: Cambria,serif; font-size: 11pt;">Lodish, H.; Berk, A.; Zipursky, SL., Purifying, Detecting, and Characterizing Proteins. //Molecular Cell Biology// **2000,** 4 (3.5). <span style="color: #222222; display: block; font-family: Cambria,serif; font-size: 11pt; text-align: justify;">[2] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J., Protein product and purification. //Nat Methods// **2008,** 5 (2): 135-146.