If you want people to give you advises, you should precise what you are looking for exactly.

The article below is an example of what we did with the venom of Conus Consors. The toxic molecules are generally peptides or proteins. We purified and characterized the sequence of one toxic peptide using HPLC and mass spectroscopy.

https://www.researchgate.net/publication/221733440_A_novel_-conopeptide_CnIIIC_exerts_potent_and_preferential_inhibition_of_NaV1.21.4_channels_and_blocks_neuronal_nicotinic_acetylcholine_receptors?ev=prf_pub

Article A novel μ-conopeptide, CnIIIC, exerts potent and preferentia...

Hi Ludovic

thanks for the link. I am trying to look at Scorpion venom of certain species which has never been worked with. So i am a little bit confused how to proceed with the venom studies. Moreover we have an analytical HPLC of waters and i am unsure if i can separate molecules using this instrument...as the injection/sample loop is of 10ul. so can you kindly tell me how to proceed now...I am completely new to the field and thus i am trying to find out some help from the experienced counterparts.

How much do you have in terms of concentration and volume?

Hi Jeffrey and Angela

Thank you first for you answers and interest in my question.

@Jefferey: As per the volume it is very less as i can only extract around 10ul or less of sample from each individual. Right now i have a final volume of 400ul and concentration of 6mg/ml.

@ Angela: Yes i was thinking of the same thing as you have mentioned Angela. But the point is my volume as i mentioned above is very less and can you explain a bit about how to fractionate them to study the activity further??

6 mg/ml is very high for most techniques. For example, Comassie staining of gels has a sensitivity of around 100 nanograms. If you load 5 uL of each sample into each lane, right now you have around 30 micrograms. You should be able to comfortably dilute your samples at least 10 times for gels and maybe 5-10 times for HPLC.

Thanks Jeffrey for the answer..but my question is still there how to fractionate.. would using gravity columns suffice the separations or i need FPLC??

Are you trying to characterize (see what proteins are there without recovering the purified fractions) or fractionate the sample (recover the proteins for further biochemical analysis)?

In any case, the first step may be to run a gel to see what you have in terms of number of components and molecular weight. If it is mostly peptides (4kDa or less) as many venoms are, then a reversed phase analytical HPLC is your best option for both characterization and purification. For proteins, analytical ion exchange or gel filtration HPLC may be your best option for purification, as proteins will likely unfold using reversed phase HPLC , while peptides are frequently too similar in size and charge to separate by ion exchange or gel filtration.

Hi Jeffrey

Thank you for the answer. I am looking at the crude sample and then i would like to perform different assays on the fractionates. One more thing the injector loop that i have is 10ul can i use this small amount to purify the components? or do i need a bigger loop size?

A bigger loop would be helpful if you want to separate the proteins from your whole sample. Otherwise you need to run several runs. But as you mentioned, your concentration is 6 mg/ml and that is already quite a lot (as also stated above by Jeffrey), I suppose injecting 10 µl of your stuff to an analytical gel filtration column (let's say the Superdex GL 10/300 coulmns by GE) should provide good separation (this is far from overloading a column). The amount of protein should be sufficient enough to be detected at A280. But the amount of absorption is also protein-dependent, naturally.

But as Jeffrey and Angela suggested, run an SDS-PAGE to see what you are dealing with and focus on the purification after that. Even better if you have access to mass spectrometric apparatus, you could cut the gel bands from the gel, make a trypsin digestion and analyze the peptides using MALDI-TOF to identify what specific proteins you have in your mixture. Don't know how well scorpion proteomes are available but at least you might discover sequence homologs this way. Also, shorter peptides can be de novo sequenced using the same approach.

Good luck.

Hi Arne

Thank you for such an elaborate answer and so many good suggestions. Well I will proceed as per all of the above (everybody's) suggestions. No i do not have any access to MALDi-TOF but i am willing to send it to some of the outsourcing companies/universities with the facilities. Moreover I even have samples of 135mg/ml but again in small quantities. Any other suggestions are also welcome and thanked.

Please follow this link. The procedures described will really help you a lot.

http://www.gbiosciences.com/PDF/Protocol/Protein_Purification_Strategy.pdf

Thanks pratima for the info

Nice information