Ingenza

23-24NOV17 I run the PNPG and PNPX assay as previous time copying exactly the same protocol as in page 10 DR 117-073. The data are in PNPG_PNPX_reaction_IRM1-13_23NOV17.xls in Ingenza-PHD folder Graphs from the PNPG and PNPX assay are on page

22NOV17 Tape Station for IRM1 -13, B99 and kit positive, negative included I followed the instructions of the Tape Station. I prepared the samples exactly as it says in the Tapestation instructions. I added a tape with the barcode outside and down (the program needs to recognise it). I open the tape station controller software 2200. I place the tubes without the lids in the places at the left, I also place some tips at the right side (using the multipipette). The ladder needs to be at position 1. Press start. Save the results and also produce a pdf report. The bands produced are as follows on page Qubit concentration calculation I calculated the concentration of the protein produced in the in vitro protein synthesis kit (in mg/ml) IRM1: 2.22 IRM2: 2.6 IRM3: 1.43 IRM4: 2.16 IRM5: 2.32 IRM6: 2.52 IRM7: 2.38 IRM8: 2.54 IRM9: 2.58 IRM10: 1.94 IRM11: 2.38 IRM12: 2.4 IRM13: 2.64 B99: 2.3 Kit negative: 2.68 Kit positive: 2.88

21NOV17 I need to calculate my IRM13 protein concentration so I used the Qubit estimation first, the Absorbance method at 280nm and the BCA method. Qubit Qubit estimated that I have 202 ng/μl Absorbance at 280nm - switch the spec on -Program Scan in the PC -Set up -Accessories -Click use cell charger, cell1 cell2 -Click Baseline, baseline correction -Cary, start 400 stop 200 -Scan control - medium -OK -OK -Click the baseline -Click zero I placed my blank (which is the flow through buffer when i was concentrating my protein down) in cell 1 and my sample in cell 2. -Click Start -Name the file -Right click on the graph, peak labels, x and y labels, apply -If the peak at 280nm is not recognised then right click on the graph, cursor mode and track My y values is 0.247512. The extinction coefficient of IRM13 (according to Expasy) is 87015. So according to Beer_Lambert law Α = ecl I can calculate the concentration c = A/el c = 0.247512/87015x1(pathlength...

20NOV17 I estimated the DNA concentration in the starting material I have. I used the Qubit for this estimation. I need to know the DNA concentration because I need to have 250ng DNA per 25μl of the  reaction (according to the manufacturer's instructions). This means that I need to have  a final concentration of 250ng    in     25μl    x         in        1μl  x = 250ngμl/25μl x = 10ng/μl So if we measure the initial DNA concentration and we know that the final reaction volume is 25 μl then its easy to figure out how much template DNA I need to add. The concentration of the DNA template was calculated with Qubit as follows in ng/μl: IRM6 : 54.8 IRM7 : 30 IRM8 : 39.4 IRM9 : 39 IRM10 : 21 IRM11 : 50.8 IRM12 : 57.4 IRM13 : 48 I followed the instructions in the invitro protein synthesis kit which says to add the solution A, then the solution B, then the required water (so t...

17NOV17 I took the dialysed IRM13 protein and concentrate it down by spinning it down in the cut off filter tube. I run it on a gel to see whether is pure. I also run the IRM12 purification fraction in case that something went wrong and I was not able to see the elution. The gel did not show anything. I run again only the IRM13 protein and the flowthrough of the concentration (I did using the cut off filters).

16NOV17 I prepared some more buffers (I used 2.5% glycerol this time just to reduce the glycerol a bit since it blocks the filters and the protein is defficult to be concentrated with the cut off filter tubes) for IRM12 and purified it. I run the gels for both IRM12 and IRM13. I could see IRM13 in the last 2 elution fractions (250mM and 350mM Imidazole) but IRM12 was no visible. So I continue with dialysing the IRM13. I made 1.5 lt of TRIS 50mM buffer with 150mM NaCl and pH adjusted in a 2lt beaker. I put the 2 elution fractions in a snakeskin bag and put it in the beaker and left it opvernight on a stirrer with a stirring stone in the cold room.

14NOV17 After searching in the yak paper for finding how much protein has been used in the assays (so I know how much protein I need myself), I found that they have not stated how much protein they use in any of their experiments. According to the gel I run using the pure IRM12 protein produced, I need to use closer Imidazole concentration steps so that the contaminant proteins will eluted in the inbetween steps before my protein will elute. I was about to repeat a small scale purification of IRM13 but I realised I dont have any CHAPS (detergent I use in the sonication buffer). Instead I have Triton (which can substitute CHAPS) according to DR 0817-073 page 116. So I repeated the sonication and purification procedure using Triton in the sonication buffer. I did a purification of IRM12 and IRM13 with Triton instead of CHAPS. I sonicate the IRM12 and IRM13. The sonication mix got very foamy because of the Triton used. The sonication bubbles (need to be formed so that the sonicat...

13NOV17 I showed the PNPG and PNPX graphs to Frank (see DR1117-073 page 10). He suggested to use a negative control as well as the positive I have used (B99). I need to repeat the experiment with a negative control (water in the in vitro kit). So after this meeting I have to : Write down the list of the assays and procedure based on the yak paper Calculate how much protein I need for the assays Do a purification of IRM13 small scale Run the in vitro protein production for IRM 6-13 and also for a black and B99 Run the TapeStation for IRM6-13 and also for IRM1 Run the PNPG and PNPX activity experiment for IRM1-13 with a blank Check where the IRM12 and 13 come from (organisms) CHECKING IRM12 PROTEIN PURITY I run a gel using sample of the pure IRM12 protein produced from the previous purification and it seems that my protein is not pure. There is a suggestion to dilute it in 10ml buffer standard A (see the purification procedure in DR 0817-073 page 116) and put 1ml of resin...

PNPG PNPX substrate activity assay aliquots of diluted enzyme 100μl of 1.25mM substrate in 50mM buffer for 5 min at 40oC or 42oC (depending on the enzyme) Quench the reaction with 100μl 1M Na2CO3 pH optimum Temperature optima Thermostability Specificity of enzymes towards different substrates PNPG PNPX cellobiose MuC Influence of metal cations, monosaccharides, and alcohols Substrate selection of degree of polymerasation Synergism with xylanase 1:1 to 10:1

09NOV17 IRM12 PURIFICATION AND CONCENTRATION MEASURMENT I purify the IRM12 by spin it down and wash it with 15ml of sodium phosphate pH 7 . At end I had 500μl of pure protein (with glycerol and maybe other contaminants). I measured the concentration of the protein in Qubit and it was 2440ng/μl (I needed a lot of dilutions to be able to measure it, it seemed the concentration was very high). I measure also the IRM12 absorbance in 280nm in the high sensitivity spectrophotometre. I diluted the protein first 1:30, then 1:4 and then 1:1 with the buffer I used to wash it (sodium phosphate pH 7).  I blank with sodium phosphate pH 7 buffer and measured the absorbance at 280nm. So after all these dilutions the absorbance number was 0,463 So the absorbance of the original protein solution would be : Afinal solution = Dilution factor x Aoriginal Afinal solution = 1/30 x 1/4 x 1/2 x  0.463 Aoriginal = 240 Afinal solution Aoriginal = 240 x 0463 Aoriginal = 111.12 A =...

08NOV17 -IRM12 PURIFICATION The gel staining overnight showed that there must be some protein in the 50mM and 150mM Imidazole lanes (see page             for the gel). I used 10kDa filter cut off tubes (pink lid) to filter down my protein. I wash the filter by fill it up with water and spin it down at 4000rpm at 4oC for 10min twice. I need to concentrate my protein down to 500μl so I am filling up the filter in the tube and spin it down to 4000rpm for 20 minutes at 4oC. Each time the run finishes, I collect the flow through, fill the filter up with more of the buffer+protein solution, shake it again to prevent any blockage on the filter and spin it again. This procedure is slow because either the protein blocks the filter or the solution contains other substances which block the filter. In any case I need to help the filter goes by either shaking up and down the tube or help pipetting up and down close to the filter. I put the tube in the cent...

- I have made 0.5 lt of 1M of MES buffer adjusted to pH 6.5 for IRM12 and 0.5lt of 1M Tris adjusted to pH 9.0 for IRM13. The pI of the proteins is IRM12: 5.64 and IMR13: 8.52 but especially for the IRM12 we used the pH 6.5 because acidic conditions might cause proteins to precipitated and make assemblies (for sonication or for non clear extracts). Its not a problem to use 1-1.5 pH more or less than the pI of the protein -I also made the A buffer and the B buffer containing either MES or Tris (depending on the protein). Everything that has Imidazole is stored in the fridge. Today I am going to purify starting with the IRM12 - EVERYTHING ON THE ICE SONICATION with MES - I thaw 2.14gr of biomass which means I need 2.14 x 5 = 10.7 ml of sonication buffer. The sonication buffer will contain CHAPS, NaCl, PMSF, benzonase and Glycerol made up to the required volume with MES 50mM (see page 116 in DR 0817 - 073) - Sonication using the large probe in 28% amplitude for 7.5 minutes (...