tag:blogger.com,1999:blog-3017140681653842942.post7248234408911402391..comments2020-11-03T19:32:06.992-08:00Comments on J.A.M.M.Y.S: Lab Techniques/LMQAJ.A.M.M.Y.Shttp://www.blogger.com/profile/17180949601509896566noreply@blogger.comBlogger14125tag:blogger.com,1999:blog-3017140681653842942.post-63047964508224989222007-07-20T22:40:00.000-07:002007-07-20T22:40:00.000-07:00Hi,Erm, i mentioned in the previous step that tryp...Hi,<BR/><BR/>Erm, i mentioned in the previous step that trypsin is added right. The incubation is to allow the trypsin to digest the proteins into peptides. The peptides will then be analysed by MALDI (Proteins would be too complex to be analysed so must digest them into peptides). <BR/><BR/>Peptides will be desalted using a solution, 0.1% Trifluoroacetic acid (TFA). I am not sure of how the TFA desalts the peptides. Will update when i find out the answer.<BR/><BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-53624518623710727452007-07-17T06:29:00.000-07:002007-07-17T06:29:00.000-07:00hi!!you mentioned that sample is then incubated fo...hi!!<BR/><BR/>you mentioned that sample is then incubated for 4-6 hrs at 37oC or 16-18 hrs at 30oC. The incubation is crucial for wat process?? the purpose to incubatE?<BR/><BR/>And also how are the peptides desalted??<BR/><BR/>Vinodhini <BR/>TGO1ALsubshttps://www.blogger.com/profile/08726497773853698048noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-21545871163229134942007-07-12T07:38:00.000-07:002007-07-12T07:38:00.000-07:00Kent!! I found out the disadvantage le. The main p...Kent!! <BR/><BR/>I found out the disadvantage le. The main problem is that hydrophobic and basic proteins will be lost because they cannot enter the gel.<BR/><BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-68316896913795329032007-07-11T07:26:00.000-07:002007-07-11T07:26:00.000-07:00IPG strip is a thin strip of gel casted on a plast...IPG strip is a thin strip of gel casted on a plastic backing. Proteins enter the gel through rehydration. There are 3 types of method to do it. Passive rehydration, active rehydration and cup loading. We will be using the passive rehydration, where the gel is placed in contact with the protein sample for at least 16 hrs and the proteins will be absorbed into the gel passively. The rehydration will be done in a tray. After the sample is loaded, the strip will be transferred to a focusing tray that is to be placed in PROTEAN IEF cell, where a voltage will be applied. <BR/><BR/>Advantages:<BR/>-Convenient because bought commercially, last time the gel has to be prepared by the user.<BR/>-The gel is supported by the plastic backing while last time, the gel is very fragile. <BR/>-Strip can be stored prior to use while last time, gel has to be casted prior to experiment.<BR/>-Higher amount of proteins can be loaded as compared to last time.<BR/>-Has a higher throughput as up to 12 11cm/17cm strips or 24 7cm strips can be run at one time, while last time, one gel can only run one sample.<BR/><BR/>Couldn't think of any disadvantages.<BR/><BR/>Agarose gel is preferred as it has a larger pore size. In the case of IPG strip, the gel used is polyacrylamide(I don't know why), which has a smaller pore size, preventing the larger proteins to enter the gel. But this can be minimised by choosing a lower gel concentration(larger pore size). <BR/><BR/>Hope this answers your questions=]<BR/><BR/>Regards,<BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-23952482164678691272007-07-10T09:09:00.000-07:002007-07-10T09:09:00.000-07:00Heyy!Your MP title sounds really cool. Hm. Seems l...Heyy!<BR/><BR/>Your MP title sounds really cool. Hm. Seems like enough questions has been asked about the machine.As for the 2D separation, I was wondering how you actually go about doing the 1st dimension of the 2D separation, using the IPG strip. <BR/><BR/>What are the advantages and disadvantages of using IPG? I heard that some larger proteins may also be lost during this process? Please enlighten, thanks!<BR/><BR/>Kent TG01Kenthttps://www.blogger.com/profile/01254696635743571323noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-20585324175884223282007-07-09T05:01:00.000-07:002007-07-09T05:01:00.000-07:00Lizzie,Basically, the machine is quite good except...Lizzie,<BR/><BR/>Basically, the machine is quite good except for one disadvantage. After cutting the gel, the cutting head will put the gel in the well. But when the cutting head pushes the gel into the well, if the force is too big, there is a possibility that the gel will bounce out from the well. The lab tech would have to sit there and check that it does not happen so its not that automated. <BR/><BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-86105140840063323662007-07-09T04:52:00.000-07:002007-07-09T04:52:00.000-07:00Sharon,The maintenance steps are quite simple. Jus...Sharon,<BR/><BR/>The maintenance steps are quite simple. Just need to ensure that the 2 bottles of MilliQ water are filled. One bottle of MilliQ water is for the cutting of gel and another bottle is for flushing of probes. When the machine is started up, must always run auto-home routine, where the machine will prime the tubings and self-calibrate. After using the machine, the plastic frame to place the gel must be washed with 70% ethanol. Cannot wash with acid because the acid will corrode the frame. That should be about it. <BR/><BR/>Hope you are doing well=] Take care~<BR/><BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-81552357083293866502007-07-08T08:34:00.000-07:002007-07-08T08:34:00.000-07:00hi mingboon,your mp sound fun.i would like to ask ...hi mingboon,<BR/><BR/>your mp sound fun.<BR/><BR/>i would like to ask is there any disadvantage or limitation for the machine, Xcise. <BR/><BR/>Lizziewe are the XiaoBianTai-7!https://www.blogger.com/profile/08103713119175158043noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-68173730467480787092007-07-08T07:19:00.000-07:002007-07-08T07:19:00.000-07:00hey ming boon,what are the maintenance steps taken...hey ming boon,<BR/>what are the maintenance steps taken for the Xcise machine?<BR/><BR/>-sharon ang.tg02royal physicianshttps://www.blogger.com/profile/17022912731511892610noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-18071592343061240882007-07-07T22:20:00.000-07:002007-07-07T22:20:00.000-07:00Yup. Because our's is research de..Destaining is d...Yup. Because our's is research de..<BR/><BR/>Destaining is done by using ammonium bicarbonate(NH4HCO3). The stain is -vely charged and will bind to the proteins that are +vely charged. NH4HCO3 will make the environment alkaline and the proteins will become neutral. Thus the stain(-ve) would not bind to the proteins anymore. Stain is removed because it will affect the anaysis of peptides in MALDI.<BR/><BR/>It depends on the situation, no particular pros and cons. Lets say if by the time i need to add the trypsin, its already 5pm. Then i would choose the 30 degrees celsius for 16-18 hours so that i can come back tml morning at about 9am to stop the digestion. If i use the 37 degrees celsius one, i would need to wait until about 9pm to stop the digeston, which is not very practical. <BR/><BR/>Good luck for your MP too and hope that your title can be comfirmed soon!<BR/><BR/>Regards,<BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-56636547032466098392007-07-07T08:01:00.000-07:002007-07-07T08:01:00.000-07:00hihi! wah so fast u started your mp, i'm still thi...hihi! wah so fast u started your mp, i'm still thinking of the title!<BR/><BR/>yah have to agree with charmaine is kinda confusing. ok the qns...<BR/>1)step 3 how and why u destain the gel? issit got to do with result interference?<BR/>2)step 5 which incubation is recommended? any pros and cons?<BR/><BR/>good to see that everyone of us have a fruitful experience. good luck for your mp! do visit my blog to check for updates =)<BR/><BR/>Chaur Lee<BR/>TG01VASTYJhttps://www.blogger.com/profile/05770144192923839431noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-89363637355496053882007-07-07T08:00:00.000-07:002007-07-07T08:00:00.000-07:00This comment has been removed by the author.VASTYJhttps://www.blogger.com/profile/05770144192923839431noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-44490756569790019562007-07-07T04:03:00.000-07:002007-07-07T04:03:00.000-07:00CharmaineNo, ZipTip is only used in step 6 to suck...Charmaine<BR/><BR/>No, ZipTip is only used in step 6 to suck up the sample for desalting. In step 8 after the spotting then the ZipTip will be removed. If Xcise is used, spotting of MALDI plate will be done by Xcise itself. However, if you do it manually (not using Xcise), you will have to use a micropipette to spot the matrix onto the plate first, and then the sample is spotted ontop of the matrix.<BR/><BR/>Regards, <BR/>Ming BoonJ.A.M.M.Y.Shttps://www.blogger.com/profile/17180949601509896566noreply@blogger.comtag:blogger.com,1999:blog-3017140681653842942.post-66734209153874499182007-07-07T02:56:00.000-07:002007-07-07T02:56:00.000-07:00Halo! I'm kinda lost. Step 7 onward rite, u mean t...Halo! <BR/>I'm kinda lost. Step 7 onward rite, u mean the ziptip is already removed from Xcise?then, the spotting of the maldi plate is done in Xcise itself?or spotting had to be done manually before loading of the plate into the machine?<BR/><BR/>charmaine yeo<BR/>TG01The Lab Freakshttps://www.blogger.com/profile/04322986402512975861noreply@blogger.com