Marine Biotechnology - Science topic

You can even isolate the cultures and do a study. Its not that difficult. In case you need my help, you can contact me

Did either of you find a commercial fucoidanase enzyme supplier?

Dear Muralidharan Velappan ,

I suggest you to provide a subject of the PhD Thesis.

Regards,

Miquel

Maurissa Sakti .. Could you share the protocol you used to get the best result or at least the good one among all protocols you tried? Maybe someone here could give a suggestion to solve the problem by modifying the solution or some step to make it a better result.

Dear Mohammad Sibtain Kadri ,

You can try Trends in Biotechnology (IF: 14.343)

Thanks

Thank you, dear Qamar Ul Islam.

I have already read this article.

But if the problems of global cycle of phosphorus and related technologies are indeed interesting for you, I would be very greatful to you, when you let me know about new research in future.

Thank you in advance.

Also I'd like to recommend this research: https://www.researchgate.net/publication/306092164.

Arsen.

I studied the fluxes between sediment and water in heavy metal in very polluted river sediments. Redox conditions and dissolved organic matter have a very important role

@Mattias Anderson. Sorry to say than orsellinic acid derivatives are colorless unless those are conjugated with other aromatic systems (dipole/quadrupoles preferably)

I would guess that one of the components of the growth medium precipitated / crystalised due to the temperature shift. I don't think that it is "organismic" growth.

Cheers, Christian

 @Bernardo Antonio Perez Da Gama sir, This work is to determine the effects of epiphytes on Rhodophytes (Specific Macroalgae). Other parameters includes micrometabolites and other macrometabolites extraction and comparison.  

To fix fish brain tissue for TEM, I came up with the following protocol.

It works very well - Fixation is optimal. 

Best results via transcardial perfusion using freshly made fixative (<1month) 

Dear Walquiria,

Please read the following text taken from a recent review article (to download the review please use the following link:

Once the cell wall is disrupted and the biomass is fully dried, the recovery of the desired product is possible. Astaxanthin is a lipophilic compound and can be dissolved in solvents and oils. There is an abundance of astaxanthin extraction methods from H. pluvialis utilizing solvents, acids, edible oils, supercritical carbon dioxide (SC-CO2) as well as microwave-assisted and enzyme-assisted approaches. Among the recovery methods used solvent extraction and supercritical carbon dioxide (SC-CO2) extraction are considered as the most efficient, compatible, and widely used methods for astaxanthin extraction from H. pluvialis. The summary of various extraction methods of astaxanthin from H. pluvialis with recent updates is presented in Table Table55 Supercritical carbon dioxide (SC- CO2) extraction has been widely used for industrial applications due to its many processing advantages. Due to low critical temperature of carbon dioxide, the SC- CO2 system can be operated at moderate temperatures, preventing the degradation of valuable substances (Machmudah et al., 2006). Several studies have reported experiments on supercritical CO2extraction for the recovery of astaxanthin from H. pluvialis. Considering astaxanthin quality as the most important criterion, supercritical CO2 extraction is the most favorable option. Supercritical CO2 provides shorter extraction time and limits the use of toxic organic solvents. By contrast to most solvents, CO2 is relatively cheap, chemically inert, non-toxic, and stable (Guedes et al., 2011). Supercritical fluid extraction has also been tested with Haematococcus, aiming at improving the extraction efficiency. For instance, supercritical carbon dioxide (SC- CO2) coupled with ethanol or vegetable oil as a co-solvent can further increase the extraction efficiency of astaxanthin (80–90%) (Nobre et al., 2006; Krichnavaruk et al., 2008). There is an array of alternative approaches that can assist astaxanthin extraction from H. pluvialis such as solvents, acids, edible oils, enzymes, or pressurized liquids (Sarada et al., 2006; Kang and Sim, 2008; In, 2009; Jaime et al., 2010; Zou et al., 2013; Dong et al., 2014) Pressurized liquid extraction has several advantages over traditional solvent extraction. PLE requires shorter time, can be automated, uses less solvent, and retains the sample in an oxygen-free and light-free environment in contrast to traditional organic solvent extraction (Jaime et al., 2010). Recently, a simple method for the direct extraction of lipids from high moisture H. pluvialis microalgae was successfully achieved using liquefied dimethyl ether (Boonnoun et al., 2014).

Hoping this will be helpful,

Rafik

Talk to Malik Nauman, he is a coral mucus specialist.

BG-11 is rich with phosphates. They precipitate with Ca2+ and Mg2+, at least. I would suggest to use so called "cold" sterilization of the stock solution using a membrane filters with pores around 0.22 um. Do not postpone that longer than 1-2 days. Some microbes feel them self well in a fridge. You can autoclave water and mix it with cold-sterilized stock solution in any proportion. That will prevent the generation of a precipitate along with minimal changes of pH because hot sterilization stimulates the escape of CO2 from the newly prepared medium. Good luck! 

Thank you  Jackson..

Yes, Ali, for sure I could done the work by myself to identify the compounds from the extracts you mention.

However, I want to skip this process (for which I do not need RG community's advices ...), while I am requesting RG community's help to have a clear response about my question.

Best regards

Robert

Dhan Bahadur:

You may also have a look at this link for insights:

Best

Syed

Hi Palla,

I can certainly understand your concern and definitely it is a valuable question before fixing your methodology. As per my understanding GSI (Gonado-Somatic Index) is a measure to understand the gonadic growth against the somatic at a given period of time. Now consider a case where the fish has too small sized gonad,  the denominator that should consider the somatic growth gets too large to attain a value and troublesome to the measure. Thus to increase the visibility of the gonad growth and keep the denominator as low as possible, the gonad weight is subtracted from the total weight. Or otherwise the other formula is valid for any kind of fish you study.

Hope this helps you.. 

Best wishes

Hi Dilyn,

yes, I worked with open-water farming of red macroalgae during a couple of years, primarily in East Africa. Using experiments and field surveys we showed how seaweed farming alters coastal ecosystem structure (e.g. diversity, species composition) and function (e.g. sediment stabilization, primary productivity, etc.). We also wrote a book chapter about the social-ecological sustainability of open-water seaweed farming, bringin in perspectives from both economy, health, ecology and environmental science. Please look through my publication list for the papers.

We have GCMS

You can send the sample to me.

Dear Shivakrishna,

After extraction using acetone or any other solvent, simply vapourize the solvent and weigh it.

Just add MeOX-Pyridine (Methoxylamine HCl - 20mg/ml dissolved in pyridine..freshly prepared) to the dried extract and incubate the mixture at 37 oC on a thermomixer for 30 mins or 1 hour (If you use heat block, intermittent shaking should do). Followed by this add MSTFA+TMCS and incubate at 37oC for 60 mins. Spin down (in case there is still some undissolved extract) and transfer the supernatant to GC-MS vials. Run GC-MS. Good Luck

Hi Stepan,

This report may be of help to you.

Tom

That is an interesting question.  Our group has studied magnetoreception in a number of aquatic animals, including salmon.  In our experience it has been difficult to come up with "easy" tests for magnetoreception because most such experiments require precise manipulation of magnetic fields, and this in turn requires carefully designed coil systems and magnetometers (for measuring the fields).

     That being said, what is needed (in principle) to demonstrate magnetoreception is just convincing evidence that the behavior of an animal changes in some way in response to different magnetic fields.  Thus, one could hypothetically use a magnet instead of a coil system to manipulate magnetic fields.  The caveat is that the fields produced by most magnets are much stronger than the magnetic field of the earth and the fields produced by magnets are not uniform in the same way that the earth's field is (instead, the field is much stronger close to the pole of the magnet).  For these reasons, it is possible that animals that detect the earth's magnetic field may not detect or respond to strong, irregular fields produced by magnets.

     Two basic approaches have been used to demonstrate magnetoreception.  One approach takes advantage of naturally occurring behavior in an animal, such as the tendency of an animal to migrate in a certain direction using earth's magnetic field for  guidance.  By manipulating magnetic fields, researchers can then demonstrate that the behavior changes when the magnetic field changes.  In salmon, this approach was used successfully in early experiments by Tom Quinn at the University of Washington, and more recently by Nathan Putman at Oregon State University.  A second approach is to try to condition (train) animals to respond to magnetic field cues by performing behavior that does not occur naturally (for example, conditioning an animal to press a paddle or approach a target in the presence of an unusual magnetic field).  This approach has been used successfully in several experiments with animals such as chickens, bees, and pigeons, but it often takes considerable time to condition the animals, so this approach is not always easier than taking advantage of spontaneously occurring behavior.

      As Joachim Pimiskern noted, a complication of experiments with magnetic fields is that changing a magnetic field also generates a transient electric field.  Salmon are not known to detect electric fields, but some fish are.  Thus, if you are working with a species that might have both magnetoreception and electroreception, then steps have to be taken to ensure that the animals are really responding to the sensory cue that is intended to elicit the response.

      I hope you are able to discover an "easy" test for magnetoreception.  That would be a valuable discovery for those interested in learning how animals detect magnetic fields -- which is still not clearly understood in any animal!

Best regards,

Ken Lohmann

I am using this composition may be it will be helpful for you

ok, I will try it again..... is my chemical composition sod periodate is right??? 65 mg sodium iodate, 7.7 gm ammonium acetate and 10 mL acetic acid..

Thank you for your tenacity.

Well, I've already replied the question "is the epiphyte relevant in driving abundance/richness of clients?" and the answer is yes. Now I'm going deeper and my questions is "are the epiphyte density (low/high - 1st factor) and the epiphyte type (living/mimic - 2nd factor) relevant?"

I collected the host (Cystophora) and some natural epiphytes, I washed them and I manually transplanted them on the host (in 0, low and high densities) and I did the same with some other host at which I attached an epiphyte mimic.

The idea is that the abundance of clients is dependent on epiphyte size and that clients may use the living epiphyte more efficiently than the mimic one. But these are just thoughts.

Replying the other doubts:

- my variables (abundance and richness) are standardised per gDW of the host... in here I'm considering the size of the host

- second, I use PERMANOVA based on resemblance matrix in Euclidean distance.

I remark that I've already run the analysis removing all the controls (with a 2x2 design then... just low/high and living/mimic) and the results don't change. I think the software, specifically created for this kind of analysis, is smart enough to make the right considerations (considering my input rules correct).

Thanks

AS

if they are free fatty acids they will be dissociated to COO-  you need to drop the pH so they are present as COOH then they will partition into hexane or other non-polar solvents.  Determine the pH you need based on the Pka of the fatty acids you are interested in.

I like using MNNG. It is a chemical mutagen but you can inactivate the chemical by tossing used materials in an acid bath for easy clean up. We have used it successfully for green algae as well as cyanobacteria. I have seen publications using it for a lot of other algae. Need to do a kill curve using the chemical to get >95% kill then treat your Tetraselmis to the 95% kill on a selective medium (preferably) and pull mutant colonies - obviously at 95% kill some will be resistant wild-type but that is why a selective medium would be best.

Be careful since it is a mutagen - gloves and properly dispose of all materials. Acid treat all equipment and reusable instruments and glassware.

Hi, i am very happy that you have found what you are looking for.

Dear Eswar,

Please let me know what your trying to extract? if your working for proteins and peptides try with Ethanaol, ACN:H2O, and Methanol:H2O.  Acetone and N-hexane are not appropriate solvents for MS. If you still facing ionisation problem add TFA to the sample. 

Good Luck

BNRao

The precipitates are calcium salts. http://www.rsc.org/learn-chemistry/resource/res00000484/chemicals-from-seawater?cmpid=CMP00004765. This is pretty common in artificial seawater. You'll also see it when you mix alcohol into seawater. 

The precipitates don't pose a problem other than causing the medium to become cloudy. You can autoclave the seawater first, then filter it, and then use it in your medium (autoclave with the agar).

I agree with most of the previous comments. The assumption for each of the indexes are not exactly the same, so, further comparisons may be problematic. Regarding methodological issues there are several approaches to meassure conditions. I recomend, among others, the following reads:  

As far I know and understand, there is no such policy existing. Once Dr. Ayyappan (DDG Fisheries of ICAR) had organised a meeting on Policy paper. But it does not really say anything. You can go through it. Available on ICAR website

The usual solution in vertebrates is carbamylcholine chloride (carbachol). It stimulates both muscarinic and nicotinic receptors and is quite stable. I don't know about its pharmacology in mussels.

Ach is insanely hygroscopic. It will absorb water in a vacuum dessicator over dessicant and becomes a sort of wet lump.

Hi Chris,

I don't know if your still looking for an answer, but we have used the protocol from Sunnucks and Hales (1996) for DNA extraction of larval fish. We were running some microsats on them. I don't know if it would be useful for eggs. Good luck.

Regards,

Andrew

Sunnucks P, Hales D (1996) Numerous transposed sequences of mitochondrial cytochrome oxidase I–II in aphids of the genus Sitobion (Hemiptera: Aphididae).

Molecular Biology and Evolution, 13: 510 – 524

thank you

Maybe you could try visible implant elastomers. Success probably depends on the type of sea cucumber, but it might be worth a shot. http://www.nmt.us/products/vie/vie.shtml

This could be Tubastrea sp belonging to Dendrophyllidae family. However, skeletal structure needs  to be studied for confirmation.

“Palmer – Maloney”is a good camera for phytoplankton. ”Sedgwick Rafter” also it can be used but must pay attention to the working distance of objectives that will work.

Hello Jackson, 

I appreciate your response, that is a great help!

Cheers,

Allison

 http://www.int-res.com/articles/meps/130/m130p295.pdf

You may also ask SWL rope lifting and testing company.(http://www.swl.org.uk/destructive-and-non-destructive-testing/)

Please refer 'Sri Lankan Seaweeds: Methodologies and field guide to the dominant species' by Coppejans et al. This can be also downloaded at http://www.abctaxa.be/downloads/volume-6-algae-sri-lanka

I regularly culture larvae of Evechinus in 20 litre plastic buckets at reasonably high density (>10/ml) for bulk samples needed for proteomics without problems such as raised above.  We use a aeration system modified from Mike Russell's one in green sea urchins to be static.

I'm sorry I haven't any work on this issue. I attached some files may be it useful for you.

Regards

Fikrat

Yes, it can be done. You can increase the success of your analysis by (i) taking multiple skin samples from different parts of the body, (ii) take the skin sample from as deep in the skin as possible (deeper = less UV damage), (iii) amplify short DNA fragments, (iv) optimize your primers for the relevant species.

If possible, try to sample other types of tissue as well.

See Luksenburg et al. (2015) for an example. We obtained DNA from samples that had been exposed to tropical heat for weeks/months and got good results.

Luksenburg, JA, Henriquez, A & Sangster, G 2015. Molecular and morphological evidence for the specific identity of Bryde’s Whales in the southern Caribbean. Marine Mammal Science 31: 1568–1579.

I should mention that it requires epifluorescence microscopy

Great! Thank you for your answers :)

Cheers

As per the literature there are various enzymes secreted by the microbes for the degradation of the plastic,  some are as:

1. Alkane hydroxylase (Pseudomonas aeroginosa) (Belllaj et al., 2006)

2. Lipases (Uchida et al., 2000; Bhardwaj et al., 2012)

3. Laccase (Rhodococcus ruber)

4. Lignin, manganese dependent peroxidase (LiP & MnP)

5. Serine hydrolasses

6. Esterases

7. Putative polyurethanases

8. Phenol oxidase (laccase)

9. Heme peroxidase (lignin peroxidase)

10.Protease (Bacillus sp. and Brevibacillus)

Dear all

I agree with Ana on the gravimetric method.  The method I published involve "direct in-situ esterification" of all types of lipids [FFA,PL and Neutral lipids" in one step [HCl;Methanol] and subsequent quantization through GLC

Let me know if you need any more than the published process

These are all epiphytes may be animals (Bryozons) or some agal forms  ( Ectocarpus and Bangiopsis) also present on the thallus of Sargassum, Laminaria and Lobophora. Before extraction of the fine chemicals we have to clean the material properly remove all debris and epiphytes and allow to shade drying for getting quality products. Thank you

You can do the extractions as described above. Another alternative is using solid phase extraction as well.

I would rather transport Kappaphyus alvarezii without seawater, but under cool conditions. This alga can tolerate desiccation of moderate proportion. Please ascertain that there is no direct contact of ice packs with alga, use plenty of thermocol pieces for this purpose, by this way there shall be less physiological damage. After transporting it to desired destination put algal material with sufficient seawater under ambient conditions and then acclimatize slowly to the culture conditions of your choice. It may take a week or two for the algal samples to start growing, although some tissue may perish.    

It all depends on what kind of experiments you are following, experiments studying physiology required different protocol. However in all the cases too much cool conditions might hamper the prospects of algal survival.  

Maybe this article/ scientific work group could help you for the identification.

A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms

J. I. Carreto, M. O. Carignan, N. G. Montoya

Dear Sukma,

I'm not sure I fully understand your rather cryptic question... If your concern arises in the context of the carbon cycle's influence on climate change, then I'd say that light is generally not a limiting factor (in the photosynthesis process), at least in the upper layers of water where algae live, so that attention has not focused on this particular aspect of the assimilation of atmospheric carbon dioxide into organic materials. However, if you are concerned about a different issue, then please clarify your question.

Cheers, Michel.

Dear Chakraborty

Check the following links

Good luck

Most of the work has been carried out on Laminariales (kelps). Iodine is mostly stored as iodide in the extracellular matrix located in the peripheral tissue (Kupper et al. 2008). Iodide has been suggested as an inorganic antioxidant. Iodine-rich kelps are good for helping the thyroid to function and the treatment of goiter. But, if you want to reduce the iodide content, an oxidative stress should release it to the atmosphere (but is that good? It has been linked to the formation of clouds).

Hi Martina,

It is possible to transplant succesfully this species. However,  the most important thing is to collect the individuals without dislodging its byssus from its attachment. If not, the individuals surely will die. The transport must be done quickly (as much as possible) and try to keep the individuals in the water. If you transport them by zodiac or similar...put the fan mussels in plastic tanks with a portable air pump. Once the individuals are in the new habitat is important to bury them for up to half of the shell ensuring that no swell or storm will affect their position. Progressively they will generate new byssus threads and together with the original ones will conform the new fixation network.

Every individual transplanted by me was reintroduced in the same original place (marked with GPS) with 100% of survival rate. However, i can not ensure if new habitat conditions will be good for them due to parameters such as currents, food availability, etc.

For any other question please don´t hesitate to contact me.

Best regards

Sergio

My work emphasizing on Synchronization's of oestrus and study the breeding cycle of Dorcas gzazelles  

Bonjour Nicolas,

ces rendements sont facilement atteints sur des biomasse non déshydratées. Pour des extractions sur poudre sèche, la valeur est plutôt deux fois moindre (~10%).

La teneur en phycocyanine et la composition des antennes PC/APC/PE ne dépend pas uniquement du C/N. C'est aussi, pour une variété donnée, une question  de quantité et de qualité de la lumière.

Au plaisir

Sea animals contain different marine toxins such as tetrodotoxin, saxitoxin, ciguatoxins and jhingerotoxins. These are important metabolites which are synthesized by fish, sea snakes and aquatic mammals to protect themselves from predators and food and feeding. Sea stars and sea cucumbers are well known to contain steroidal saponins as the toxic substance. Toxins can be extracted in alcohols by homogenization and centrifugation then pass saponin-like agent by a dialysis or gelfiltrations on Sephadex G-10, G-25 and Bio Gel P-4.Some high molecular

substances like proteins are also reported from sea urchins to be toxic which can be separated by Trichloroacetic acid precipitation or in EDTA based Phosphate buffer slaine pH 6.7.

Growing algae will increase their size with a rate depending on light amount and quality, temperature and amount of nutrients available. When they reach so called treshold size they will become able to undergo cell division, a process called attainment of commmitment point, but it will take some time before they actually divide. So the treshold size at commitment point is different from actual size at cell division if the cells are grown on light while it will be similar if they are moved into dark at the moment of commitment point. If you want to measure a treshold cell size at commitment (which is usually not affected by light or nutrients but is affected by temperature) you can simply grow the cells on continuous ligth until certain cell density is reached, then treat them with prolonged dark (24 hours) and then measure cell size by for example Coulter counter. The maximum cell size reached by these cell is a rough estimate of commitment treshold cell size since all the cells above the treshold size will divide in dark. There are more precise measurements but this estimate is usually very good and sufficient. 

I fear I may be exposing my ignorance, but as an alga, I think Kappaphycus contains carrageen, while animal parts such as shark fins contain collagen. Both have a gelatinous nature, but are they the same substance?

I agree with Roshni.  I have used that kit in the past to sequence DNA from rat kidney.  It works well, is easy to use, and is not very expensive.

Zobell marine broth 2216 contains ferric citrate and sodium sulfate. In the presence of sulfur reducing bacteria in the marine sediments, concurrent reduction of ferric and sulfate to ferrous sulfide.

   

from my experience, microalgae need some time for adaptation to the new setting (culture medium, cultivation condition and cultivation mode). i usually put more than the initial seeding that i normally use which is 10%. it depence on the adaptation capacity of the microalgae to the PBR/open pond. last time, i used 30% of initial seeding to adapt spirulina in waste water. because it takes too long for the adaptation process. after the adaptation process, i used 10% of initial seeding for my original experiment. so, u can modify the initial seeding based on the adaptation process of the microalgae....

Collect seeweed wash them carefully and dry in shade. Make its powder in powered mixy then dissolve in the solvent. Then partition it in low to high polar solvent system for partitioning to collect fraction of your choice by changing the polarity. 

Can you purify that larger band and sequence it? Then you will know what‘s that.

The non-specific amplification happens very often in ITS-PCR. Usually the template amount of colony PCR is hard to quantify and sometimes exceeds the optimistic concentration, which will probably lead to the non-specific amplification. 

In your case, as long as you are getting the results you want, you don't really have to optimize the PCR conditions.

While metagenomics would of course give you much more and better information about your communities, it still has the same problem as DGGE in terms of PCR bias. So, the choice of primer will influence your results independent if you use DGGE or metagenomic sequencing.

Depending of what you want to get out of your analysis, my suggestion would be to do the analysis with two or more different 16S primers to reduce the influence of PCR bias and see if they lead to similar results. A good resource for 16S primers that can be used for various applications and their coverage can be found in the paper

Nucleic Acids Res. 2013 Jan 7;41(1):e1. doi: 10.1093/nar/gks808. Epub 2012 Aug 28.

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.

Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO.

Good luck with your analysis.

You may wish to consult the following paper:

Casadevall, M., Matallanas, J. & Bartolí, T. 1994. Feeding habits of Ophichthus rufus (Anguilliformes, Ophichthidae) in the western Mediterranean. Cybium 18 (4): 431-440.

I could send you a pdf if you like.

Just amino acid sequence!!!!!!Thanks_Abdallah Samy

I observed a few sections of our seaweed. With the information you have provided, I think it is indeed Solieria chordalis. Please see the picture below.

We could eventually be interested in molecular analyses. I'll let my supervisors (N. Bourgougnon and G. Bedoux, I think you met them at XXIst ISS and ISAP) contacting you.

Thank you again for your help.

Regards,

Kevin

Actually speaking you do not get molecular markers specifically made for C-PAGE. However, you can make your own set of markers, using the primary standards of HPLC or HPTLC, but then you must decide what to mix and what not. It is a trial and error method, and may consume some of your precious time for a particular condions of C-PAGE.

Enzymatic digestion is very convenient if you are looking for peptides. On the contrary, isoelectric précipitation will provide proteins instead of peptides.

Many commercial enzymes are available for the enzymatic solubilisation of proteins . If you are interested in preparation of hydrolysates with bioactive peptides, whatever the quantity of final hydrolysate, it is very important to control the enzymatic hydrolysis, and for that purpose, the pH-stat method combined with size exclusion chromatography  (to estimate the molecular weight of peptidic populations) are the best one. According to the enzyme chosen, and consequently, the pH for hydrolysis, you should use (or not) the pH-stat method ( for pH above 7.5; please see the publications of Adler-Nissen). For starting hydrolysis if you are not familiar with this method, may i suggest the use of Alcalase 2,4L from Novozymes (pH 8; temperature :50°C; E/S ratio ranging from 0.5 to 3%).  Good luck!

Hi,

Maybe this could be also interesting.

Hi Vijay,

Broadly, the steps involved in isolation of proteins and peptides from a food source would be size reduction, defatting, removal of phytochemicals using solvents, and extraction of the soluble proteinaceous material by using an alkaline medium (NaOH and Borate-based buffers or pure water is commonly used). This is followed by centrifugation. Another commonly used method involves isoelectric point-based or salt-based precipitation of proteins. Attached here are links to two papers with details of protein extraction, isolation and purification that may be of help to you. Best of luck!

-lakshmi

Pooling more than one animal for each sample will minimize the effects of biological variation between individuals. I did this recently for a microarray experiment. However, Kenneth is right that this does not mean you have an n3 simply by pooling three. You would need at least 3x3 controls and 3x3 tests.

I would like to recommend to use Earls buffer

Try to use pure solvents like 100% acetone. The water can be interfering somehow in the absorbance. Otherwise if you can manage to do a spectra of a known concentration solution in 100% acetone from 350-750 nm at 1 nm steps and send me the absorbance values at each 0.5 nm I can apply an algorithm to it and confirm your concentration. Cheers

I recommend taking a look at the Halo-handbook. It's a free download online, and a great reference for halophile research! ;)