BioForum Method Discussion http://www.protocol-online.org/forums Sun, 19 May 2013 17:48:47 +0000 60 Oxygen use in bacteria http://www.protocol-online.org/forums/topic/29325-oxygen-use-in-bacteria/
I used anaerobic bags to test if my bacterial isolates could grow in the absence of oxygen. There was clearly visible, hence little growth. In contrast, when incubating aerobically strong growth is observed. Thus, I would consider the bacteria as facultatively anerobic.

As negative control I used Micrococcus luteus which did not show any growth.

But: My positive control (Staphylococcus saprophyticus, facultatively anerobic) did not grow/very very little in the anaerobic bag. I am confused because I expected them to grow. I do not have a strictly anerobic bacterium. I do not have anaerobic indicator strips.

The questions I would like to ask is whether this method is adequate in judging a species as facultatively anaerobic and why S. saprophyticus did not grow. I inoculated from fresh cultures on appropriate medium.

Furthermore, does anybody know for which purpose oxygen is metabolized in anaerobic spp.? From this metabolization H2O2 is produced which cannot be detoxified. But why do they use oxygen?



Thanks a lot!]]> Sun, 19 May 2013 17:48:47 +0000 http://www.protocol-online.org/forums/topic/29325-oxygen-use-in-bacteria/ methods to extract RNA from blood sample? http://www.protocol-online.org/forums/topic/29320-methods-to-extract-rna-from-blood-sample/ i have tried trizol method and some company kits but didnt get good results.]]> Sun, 19 May 2013 09:38:03 +0000 http://www.protocol-online.org/forums/topic/29320-methods-to-extract-rna-from-blood-sample/ GHR gene location of water buffalo? http://www.protocol-online.org/forums/topic/29319-ghr-gene-location-of-water-buffalo/ i need to know that in bovine and water buffalo the disorders and diseases related to growth hormone receptor (GHR) or growth hormone binding protein (GHBP)?
also the gene location of GHR of water buffalo with refrences.
pleases can anybody help me out?​]]> Sun, 19 May 2013 09:33:09 +0000 http://www.protocol-online.org/forums/topic/29319-ghr-gene-location-of-water-buffalo/ Western Blot - Quantifying Tumor samples from two different blots http://www.protocol-online.org/forums/topic/29318-western-blot-quantifying-tumor-samples-from-two-different-blots/
I'm new here. And I have a question regarding about quantifying bands on two separate membranes.

So I have a total of 13 protein lysate samples from tumors. Out of those 13, there are 7 control samples, and 6 treated samples.

We only have pre-casted 10 lane gels, so I loaded those 13 samples on two separate gels.

gel 1 has 3 control + 3 treated samples

gel 2 has 4 control + 3 treated samples.

I'd like to ultimately compare the treated samples to control.

After normalizing to the loading control (alpha tubulin), how can i properly combine the numbers from each blot so that I'd have 7 data points for the control group and 6 data points from the treated group? As of now, the trend between the blots between control and treated is consistent (blot 1 and 2 - ~40% decrease of treated compare to control,) but the raw value varies from blot 1 to blot 2, so significance was not reached when entered into graph pad prism.

I'm hesistant to rerun the samples on a gel with more than 13 lanes so i can run all the samples together because we have little of the lysates.

Any help would be appreciated!]]> Sat, 18 May 2013 23:42:56 +0000 http://www.protocol-online.org/forums/topic/29318-western-blot-quantifying-tumor-samples-from-two-different-blots/ SH-SY5Y and Poly-L-Lysine http://www.protocol-online.org/forums/topic/29316-sh-sy5y-and-poly-l-lysine/ I am having problem with neuroblastoma cell line, in particular when I seed the cells on the petri dish, most of them remain in suspension.
Looking on line I see that to overcome this problem some people use poly-l-lysine but I have no idea how to use it,
furthermore, sigma has many type of poly-lysine, could please someone tell me how to use it ? and which kind of poly-lysine is good in term of molecular weight ?

Regards

Patrizio]]> Sat, 18 May 2013 01:21:30 +0000 http://www.protocol-online.org/forums/topic/29316-sh-sy5y-and-poly-l-lysine/ classic cloning with restriction enzymes into empty pcDNA3.1D http://www.protocol-online.org/forums/topic/29314-classic-cloning-with-restriction-enzymes-into-empty-pcdna31d/
Thanks a lot!]]> Fri, 17 May 2013 16:13:34 +0000 http://www.protocol-online.org/forums/topic/29314-classic-cloning-with-restriction-enzymes-into-empty-pcdna31d/ Protein folding in different species http://www.protocol-online.org/forums/topic/29313-protein-folding-in-different-species/
I've overexpressed a protein in both bacteria and human cells (293T). This protein is immunomodulatory and can inhibit TNFalpha secretion of immune cells. So I designed a wildtype and also mutated form that should be expressed by both species.

Now I have this curious findings:
The WT protein from both bacteria and 293T is working well in-vitro (positve effect).
The MUT protein from 293T cells has no effect at all (that's alright as it should serve as negative control).

BUT: The MUT protein from bacteria is also able to abrogate TNFalpha secretion (which should not be the case!).

I've confirmed the sequences of the vectors that have been used for transformation and they were okay. Also the isolated plasmid from bacteria carries the mutation and is fine.
Furthermore the MUT protein from 293T is recognized by ELISA, but NOT the MUT protein from bacteria (although they should be the same). So I'm wondering whether the folding of the MUT protein is different in human 293T and bacterial cells?! Or what explanation would you give for this fact?

I'd be grateful for some advice...
Regards :)]]> Fri, 17 May 2013 14:36:58 +0000 http://www.protocol-online.org/forums/topic/29313-protein-folding-in-different-species/ Immunoprecipitation- Incubation time with beads http://www.protocol-online.org/forums/topic/29312-immunoprecipitation-incubation-time-with-beads/
In all the protocols I see it is written to incubate the Lysate solution with the antibody overnight and then add the beads next day for an hour or so. I made a mistake and add the beads last night. They are incubated with AB and sample all over the night. Is it going  to be a problem? Am I going to have a huge background? should I increase the wash? I pre-cleared the sample with Rabbit serum and beads before.

Please help me
Thanks]]> Fri, 17 May 2013 13:36:30 +0000 http://www.protocol-online.org/forums/topic/29312-immunoprecipitation-incubation-time-with-beads/ experiments related to epigenetics http://www.protocol-online.org/forums/topic/29311-experiments-related-to-epigenetics/ At the moment I have a BS degree and would like to perhaps pursue more education in the future studying Epigenetics, specifically gene expression resulting in chronic low dose exposure to environmental elements. Its been an interest for years, after hanging out with many chemophobic individuals, and studying toxicology while pursuing my degree.
However, I have NO LAB SKILLS.  I have read more protocols than I can count but have no idea where to get started.  I was going to start with simple yeast and perhaps get my hands on some that has been modified and go from there. However other than culture techniques and extractions I am clueless after that point.  
If you guys have any general experiments I can run to gain some skills please share them.

Thanks.]]> Fri, 17 May 2013 13:09:10 +0000 http://www.protocol-online.org/forums/topic/29311-experiments-related-to-epigenetics/ Debris in cell culture http://www.protocol-online.org/forums/topic/29310-debris-in-cell-culture/
Thanks to everyone!

-Lara-]]> Fri, 17 May 2013 13:01:09 +0000 http://www.protocol-online.org/forums/topic/29310-debris-in-cell-culture/ What housekeeper to use with HEK cells? http://www.protocol-online.org/forums/topic/29308-what-housekeeper-to-use-with-hek-cells/
I am working with HEK293 cells and tried to check my RNA integration by using usual housekeepers (EF-1a, GAPDH, SDHD) but apparently they are not able to bind to this template, although in silico analyses revealed that they should. I tried them with quantitative PCR as well as with conventional RT-PCR with the same results.

I would be really happy for any suggestions.

Thanks a lot,

Best
Stephanie]]> Fri, 17 May 2013 11:39:41 +0000 http://www.protocol-online.org/forums/topic/29308-what-housekeeper-to-use-with-hek-cells/ Housekeeper for HEK cells? http://www.protocol-online.org/forums/topic/29307-housekeeper-for-hek-cells/
I am working with HEK293 cells and tried to check my RNA integration by using usual housekeepers (EF-1a, GAPDH, SDHD) but apparently they are not able to bind to this template, although in silico analyses revealed that they should. I tried them with quantitative PCR as well as with conventional RT-PCR with the same results.

I would be really happy for any suggestions.

Thanks a lot,

Best
Stephanie]]> Fri, 17 May 2013 11:38:29 +0000 http://www.protocol-online.org/forums/topic/29307-housekeeper-for-hek-cells/ Desalting resin http://www.protocol-online.org/forums/topic/29306-desalting-resin/
we would be packing them in XK 16/40 column and use for desalting so what amount of sample can be desalted at a time.

kindly suggest

thanks

Posted Image]]> Fri, 17 May 2013 11:26:00 +0000 http://www.protocol-online.org/forums/topic/29306-desalting-resin/ desalting resin http://www.protocol-online.org/forums/topic/29305-desalting-resin/
we would be packing them in XK 16/40 column and use for desalting so what amount of sample can be desalted at a time.

kindly suggest

thanks]]> Fri, 17 May 2013 11:17:53 +0000 http://www.protocol-online.org/forums/topic/29305-desalting-resin/ Negative control antibodies (Anti-mouse C5 Clone BB5.1) http://www.protocol-online.org/forums/topic/29303-negative-control-antibodies-anti-mouse-c5-clone-bb51/
Can I use the negative control Biotinylated Anti-Mouse IgG1(raised in horse against mouse) antibody as a negative control?

Many thanks]]> Fri, 17 May 2013 05:59:39 +0000 http://www.protocol-online.org/forums/topic/29303-negative-control-antibodies-anti-mouse-c5-clone-bb51/ Type I Collagen Vs. Poly-L-lysine Vs. Gelatin Coated coverslips (NSC34 cells) http://www.protocol-online.org/forums/topic/29302-type-i-collagen-vs-poly-l-lysine-vs-gelatin-coated-coverslips-nsc34-cells/ I just had a quick question about culture substrates.

My supervisor wants quantitative confirmation that my cells are doing better on poly-L or collagen than gelatine before he coughs up $284 for pure type I rat tail collagen (from Sigma) as he does not have the funds. I wanted to know whether or not that was possible? By that I mean, I'm not sure whether that sort of data would be quantitative (e.g. cell density) or qualitative (cell adherence) because NSC34s will happily proliferate whether or not they are properly adhered to the surface (they are a reasonably aggressive proliferating cell line).

Please let me know what you think. Is it more of a qualitative or quantitative measure?

And which substrate would be best? Gelatin (which I've heard dissolves in modern day media over a period of 1 week) or collagen (at 0.01%) or poly-L (at 0.01%)?

Many thanks!]]> Fri, 17 May 2013 04:52:59 +0000 http://www.protocol-online.org/forums/topic/29302-type-i-collagen-vs-poly-l-lysine-vs-gelatin-coated-coverslips-nsc34-cells/ Deactivation of bacteria for competition study http://www.protocol-online.org/forums/topic/29301-deactivation-of-bacteria-for-competition-study/
I'm doing fungal fermentation. Every now and then I do have some contamination but it wasn't serious. I didn't use any antibiotics as my fungus is usually strong enough to overcome the contamination (I do practice sterile techniques)

I've been only analysing the one metabolite produce by the fungus (metabolite A). Only recently I was able to analyse another metabolite that was said to increase as metabolite A increases (metabolite B )

I was surprise to see how random the production would be compared to metabolite A. After some time, I suspect metabolite B was being produced as a response to the contamination, hence that is why the production is so random even within the same replicates (while metabolite A was so stable in production)

I already cultured the contaminating bacteria. I'm thinking of introduce them into my fungus culture in 2 different conditions - live and deactivated (like vaccine). Does anyone in this forum has experience in deactivating bacteria for this kind of study? I afraid heat would change the conformation and chemical would be too poisonous to my fungal culture.]]> Fri, 17 May 2013 01:51:40 +0000 http://www.protocol-online.org/forums/topic/29301-deactivation-of-bacteria-for-competition-study/ How to prepare serial dilutions of RNA or cDNA http://www.protocol-online.org/forums/topic/29300-how-to-prepare-serial-dilutions-of-rna-or-cdna/

Attached Thumbnails

  • Amplification Plot.jpg
]]> Thu, 16 May 2013 20:51:37 +0000 http://www.protocol-online.org/forums/topic/29300-how-to-prepare-serial-dilutions-of-rna-or-cdna/ RNA extraction by trizol http://www.protocol-online.org/forums/topic/29299-rna-extraction-by-trizol/ If someone is doing RNA extraction by Trizol method Can the the protein pellet that is obtained be used for expression analysis or this method waste the cellular proteins. I wanted the protein pellet for doing SDSPAGE and 2D

Regards,
Roshan]]> Thu, 16 May 2013 20:13:14 +0000 http://www.protocol-online.org/forums/topic/29299-rna-extraction-by-trizol/ Problems in expression of target protein in E. coli http://www.protocol-online.org/forums/topic/29298-problems-in-expression-of-target-protein-in-e-coli/ I'm facing a problem in expressing my target protein by using BLR(DE3) E. coli  for months so your comments would be very valuable for me.
More than 3 years ago, I have constructed a plasmid using pET22b(+) host strain and stored in glycerol stock at -80 C conditions. The expression of my target protein had been well until last year when it began significantly decreasing in yield (like less than 10 times compared to before with the same protocol). Gradually in later expressions, I didn't get the band of the target protein when running SDS-PAGE. Seemed like E. coli expressed a different kind of protein due to it migrated at different location.
My target protein's molecular weight is 14 kDa and the unwanted protein is about 16 kDa.

So, I transformed previously constructed plasmids into BLR(DE3). And the results were the same. Strangely, the band has similar molecular weight with the unwanted protein above. I mean its molecular weight is 16 kDa.

And I constructed again a new plasmid, tested by sequencing to make sure everything is OK, transformed into a newly bought BLR(DE3) and it happened the same.

Btw,  I'm using TB solution containing extra proline for nutrients. My target protein is highly hydrophobic, contains many repetitive sequences and a His-tag at the C-terminus. I purified by using Ni-NTA resin. After purification, it always contains an impurities which molecular weight is about 31 kDa besides the target. However, these impurities precipitate during dialysis and easily removed. Recently, amount of the impurities increase and hard to precipitate so this is also another problem.

I didn't get what is wrong here? I guess the problem is not on the E.coli but the expression media? Or sth contaminant?
Anyone knows this situation before?
I'm looking forward to your advice.
Sorry for my bad English and Thank you very much (from Japan)

P/s: I just use E.coli to express the target protein so I have very little background on this field (protein expression)]]> Thu, 16 May 2013 17:21:15 +0000 http://www.protocol-online.org/forums/topic/29298-problems-in-expression-of-target-protein-in-e-coli/