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zienpiggie's Content

There have been 45 items by zienpiggie (Search limited from 22-February 19)



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#143306 confusing Western results

Posted by zienpiggie on 13 October 2012 - 10:42 AM in SDS-PAGE and Western Blotting

Hi there, I am just wondering of why were you normalizing for lipid droplet concentration? Wouldn't you need to normalize for protein content instead? I think your idea that the lipid droplet might affect the result is a possibility. The SDS Page is a highly charged system so my guess is that having something hydrophobic could lead to your samples to float/pull away the protein that is supposed to run along with the system. To be sure, I am just wondering if you could perhaps defat your samples so that you will only be running the protein fraction that was previously associated with the lipid fraction?



#136039 Significant results are hard to come by

Posted by zienpiggie on 16 June 2012 - 01:58 PM in Venting and Counseling

Yes it can turn the way you described in your post. The past few years have been very grueling for me too. I think that part of the PhD training is also knowing of when it is time to switch direction and change strategy. In the end we just have to sit in front of our data, look at it and accept what it is saying and deal with it accordingly, including finding new leads that you can pursue. If the problem is that your research question is too big, then this is the time that you decide for yourself to narrow it down and focus it so that you can finish in a timely manner.

I hope it helps. Don't give up :D.



#133614 Primary cell line

Posted by zienpiggie on 26 April 2012 - 04:15 PM in Tissue and Cell Culture

Thanks Bob1. I probably should pick up Freshney to refresh. Thanks for the confirmation!!



#133584 Primary cell line

Posted by zienpiggie on 26 April 2012 - 10:09 AM in Tissue and Cell Culture

Hi all,

I bought a cell line derived from normal cells/nonmalignant cells from atcc, and it's been suggested in the literature that the cells are primary cells. I however met someone who argued that it's not really a primary cells because 1. it is not derived directly from the donor and the person said that primary cells would usually last only for one passage or so and that's it. 2. they are transformed because I can passage them up to 8 passages.

Also, some suggests that primary cells should never be freeze thawed upon arrival and be used within five passages?



#132096 Is it possible to assay the carbohydrate profile of a cell?

Posted by zienpiggie on 30 March 2012 - 03:16 PM in Biochemistry

Just a thought.. how about hplc-coupled with refractive index detection or you probably can identify those sugars with mass spec?



#132095 Western blot mystery - possibly a sample preparation problem?

Posted by zienpiggie on 30 March 2012 - 02:48 PM in SDS-PAGE and Western Blotting

Also by any chance, is your gel assembly tight? or if they are made fresh? If they are dried out, the gels shrink and may become detached from the plate then your samples could be seeping out of the gel instead of running down. Nevertheless, it sounds like you are not getting much protein in the 25 kda to begin with. I think increasing the protein amount to load with fresh SDS probably the next thing to try.



#132094 Question about the amount of mRNA and secreted protein level!!! THX~

Posted by zienpiggie on 30 March 2012 - 02:42 PM in General Biology Discussion

My experience is that even if the genes are expressed at relatively high level, it does not necessarily relate with high protein secretion.



#132027 acceptable variation for biological replicate

Posted by zienpiggie on 29 March 2012 - 03:02 PM in PCR, RT-PCR and Real-Time PCR

Thanks Trof!



#131980 Western blot mystery - possibly a sample preparation problem?

Posted by zienpiggie on 29 March 2012 - 09:48 AM in SDS-PAGE and Western Blotting

When you commassie your gel, can you see distinct protein bands on your samples around the 25 kda? Also, may be you could ponceau your membrane to make sure you have a decent transfer. I also don't fully understand what you mean by the bromophenol blue becomes barely visible within 20 minutes?



#131934 Western blot mystery - possibly a sample preparation problem?

Posted by zienpiggie on 28 March 2012 - 08:16 PM in SDS-PAGE and Western Blotting

Perhaps you can try loading more than 20 ug of protein? If you could lyse your cells with smaller lysis buffer volume, that should concentrate your protein and you can keep using low loading volume. You also didn't mention what %of gel did you use the first time around? If you use higher concentration gel (may be 12 to 15%), you could slow down the run, and then may be you could also try stopping the run before your loading marker ran out of gel.



#131904 acceptable variation for biological replicate

Posted by zienpiggie on 28 March 2012 - 12:04 PM in PCR, RT-PCR and Real-Time PCR

Hi all,

I am just wondering of what would be the acceptable level of variation for biological replicate in real time PCR. I did my statistic on 'fold change' which is derived from the delta delta ct for about 18 genes per treatment group. Some of my gene have low variation and some have high CV (greater than 20%). is that normal? The ct values for the housekeeping gene itself was not significantly different across treatment.



#131756 Determining whether media contains serum or not?

Posted by zienpiggie on 26 March 2012 - 12:11 PM in Tissue and Cell Culture

Perhaps you can try take a sample and run media that you know that hasn't been added with serum and your unknown media with protein content assay. Media containing serum turns positive in Bradford assay instantly.



#131633 normalization to protein content or cell number

Posted by zienpiggie on 23 March 2012 - 02:38 PM in Cell Biology

Thank you Bob1!



#131511 normalization to protein content or cell number

Posted by zienpiggie on 22 March 2012 - 09:26 AM in Cell Biology

Hi everyone,

I need to determine the amount of my compound in my treated cells, and I realize that some express their compounds being normalized to protein level or per cell number (e.g. per a million cell). I am wondering if there is a reason to favour one over the other? I am leaning towards expressing it per protein just because expressing per cell number by cell counting might probably give a greater variation as opposed to using bradford assay. But then I'd like to know if anyone else have insights regarding the topic.



#131157 Cell culture shared with yeast?

Posted by zienpiggie on 17 March 2012 - 12:18 AM in Tissue and Cell Culture

no I don't think you're over reacting. I almost had the same situation where my boss has microbiology-related project. I made a stern statement to my PI that I did not want any bacteria-related stuff in the cell culture area. My biggest worry is bacteria contamination due to poor aseptic technique or other blunders. If one is super careful then may be things might turn alright, but people make mistakes esp. when they are newly trained or when tired. I don't think therefore that it's worth the risk. If the cell line is something you can buy then it will cost a couple of hundreds to get a fresh start up and at least few weeks before you can have enough cells to generate data. If the cell line has unique characteristic that you/others developed and can't be bought commercially and it gets contaminated then research comes to a potentially long halt. so.. I agree with your position.. I think you need to stand your ground.



#130560 Western transfer tank heating up

Posted by zienpiggie on 06 March 2012 - 10:30 PM in SDS-PAGE and Western Blotting

what volt/ how long? It can get pretty warm at the end of the run (I could run up to 2 hours) at room temperature.



#130543 protein extraction from transwell inserts

Posted by zienpiggie on 06 March 2012 - 02:36 PM in Tissue and Cell Culture

Or perhaps you can wash twice with PBS, scrape first, transfer cells to a centrifuge tube or some other container then add lysis buffer .



#128107 sublimation of frozen aliquots on storage

Posted by zienpiggie on 31 January 2012 - 12:17 PM in General Lab Techniques

just curious, do you use a frost free freezer? I think the frost free freezer has a freeze thaw cycle to prevent frost from build up, but not great to prevent freeze thaw cycle from samples. That might be why your samples sublime?



#125968 Nuclear extract problems

Posted by zienpiggie on 24 December 2011 - 12:03 AM in Cell Biology

2 ug/mL is indeed really small.. I wonder what is the volume of your resuspension buffer to lyse your nuclear extract? I wonder if you are using hypertonic buffer to lyse your nuclear extract? 3



#125502 nuclear extraction question

Posted by zienpiggie on 15 December 2011 - 12:15 AM in Protein and Proteomics

Thanks Bob1.



#125348 centrifugation speed/cell prep for flow

Posted by zienpiggie on 12 December 2011 - 09:34 PM in Flow Cytometry

Hi science noob,

you are right, I made a mistake recalling what I did. It was fixed in 1% paraformaldehyde on ice for 1 hour, and then after washing with PBS twice was then placed in 70% ethanol and then stored at -20C overnight.

Your flow tubes are glass? Mine looks like it's made of polystyrene. My manual suggests to do my cell prep in the 12 x 75 mm test tubes as the 'polystyrene' tubes would have built up static so cells my adhere to the walls or something. But it does not specifically suggest 'glass'. In that case may be I should prep my samples in the polypropylene microfuge tubes instead then and only move it to the flow tubes on the last stage. Thanks for the suggestion.



#125346 centrifugation speed/cell prep for flow

Posted by zienpiggie on 12 December 2011 - 08:52 PM in Flow Cytometry

Hi all,

I am just wondering if there is a particular significance when prepping cells for flowcytometry to use relatively low centrifugation speed to spin cells down (usually at 300g)? The reason i ask is because I find that I am losing cells over the many times I need to wash/rinse cells centrifuge and aspirate supernatant. I fixed my cells in 1% paraformaldehyde overnight at -20C first and then processing for TUNEL assay. I wonder then if the problem because I did not fix it properly or was it because the centrifugation speed was just not high enough? If anyone has any insight, I'd really appreciate your input!



#125324 nuclear extraction question

Posted by zienpiggie on 12 December 2011 - 12:15 PM in Protein and Proteomics

Hi Bob1, the reason I ask is because I have often done this since the next nuclear lysis protocol for me involves 40 more minutes of lysis with vortex every 10 minutes. My application is to separate transcription factors that may shuttle between cytoplasmic and nuclear extract. I also usually add another PBS rinse to my nuclear extract to ensure that I remove my cytosolic fraction before freezing. I hope this is ok?



#125323 Performing MTT assay in suspension cells (new in the subject)

Posted by zienpiggie on 12 December 2011 - 12:08 PM in MTT, Proliferation and Cytotoxicity Assay

Hi Ana G,

Once you added your MTT solution to allow the crystal formation, you do not need to remove the MTT solution. Just add SDS/HCl straight to the wells. What you may need to remove is your treatment solution prior to addition of MTT solution, since if they are combined together, they will interfere with the MTT color development resulting in a more purplish color. You can however test if your compound will interfere with MTT assay by mixing your test compound + MTT solution in the absence of cells. If they still turn purple as opposed to control, then you have interference. If this is the case, and there is no way that you can remove the test compound and rinse your cells before adding MTT, then may be looking into other viability assay/proliferation assay is a good idea.



#125272 nuclear extraction question

Posted by zienpiggie on 11 December 2011 - 04:34 PM in Protein and Proteomics

Hi Bob1,

I wonder of why a clean nuclear prep won't be obtained when the nuclear pellet is being frozen after the cytoplasmic fraction was removed? I can see that no separation will be happening if one freeze the cell pellet without prior removal of the cytoplasmic extract. But if the cytoplasmic extract are already removed, then the nuclear extract will likely be already separated at this stage, and just need to be lysed by the hypertonic buffer, thus it will be fine if they are frozen at this stage?




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