1. What is the concentration of protein in the sampling buffer mixture which you have run??

The concentration should be atleast 5-10 microgram per well of Gel. Load around 20 micro liters per well.

2. Secondly there might be some errors while loading the sample.

3. The lyophilised samples should dissolve completely when you dilute them before the loading otherwise they will not run.

4. Try using both the markers LMW and HMW as you might not be knowing the molecular weight. It might be a possibility that you are using LMW and protein is of high molecular weight so it wont run or you might be using HMW and protein is of low Molecular weight and it outruns the gel.

5. Only proteins doesnt show absorbance at 280 nm. so make sure that they are proteins first by using some protein microassays.

Try silver staining, it will give better results and better staining profile.

I appreciate your kind understanding. I should it is protein as the sample loaded into HPLC was run on SDS-PAGE and it showed a band of approximately 21.5 kDa. This was the peak fraction from gel filtration chromatography. But i still want to know what are the other compounds that show absorbance at 280 nm. Do you have a idea. May be mass spectrometry identification will help furthur. Also once i lyophilize, i can visualise the white powder sticking to the walls of the container. Still i have to double-check it. Please suggest.

Hi Dr Bharat.

You could try a 15% SDS-PAGE so you can get a better vizualization of small proteins/peptides, obtaining a resolution most appropriate for your samples.

Another point that have already been cited by DrJadhavis is the solubilization of your samples. Make sure it is happening.

I work with peptides from tick's eggs and I also use RP-HPLC (C18) to isolate the fractions I need, but after HPLC I will analyse the fractions using electrospray ionization mass spectrometry. Depending on what was the objective of your research it would be a better method.

Thanks for your reply Dr. Patricia,

I did MALDI-MS and it confirmed that the C18 peaks are not proteins but may be some other compounds (may be i guess phenolic compounds). I am able to get a major band of 21.5kDa after Gel chromatography, but the same sample resolved through RP-HPLC didn't produce any protein peaks. Can anybody suggest the real problem. As iam getting a single major band through GFC, i will send it for MALDI analysis and possible N-terminal sequencing. Also please let me know how to identify the peaks that are not proteins. Please suggest.

Hi Dr. Bharat.

In a research involving peptides, reported by Esteves et al. (2009) the absorbance was measured at 225 nm. According to what you have written you are analysing at 280 nm. I belive if you change the absorbance value maybe you would be able to find a peptide/protein peak.

Unfortunatly I don't know how to identify the peaks that are not proteins.

I wish sucessfull research.

My best regards.

Thanks again.

It seems that acetonitrile have degraded my protein, that is why iam getting peptide peaks. I was looking for a protein of size 21.5 kDa from HPLC. I can get the protein through my GFC experiments (absorbance at 280nm) but not after HPLC. Also i used a C18 column but reports of HPLC purified proteins with C18 column is known. I am not able to get the protein band or peak after HPLC separation.

I will be soon doing MALDI and Edman sequencing of my bioactive protein from gel and PVDF membrane from my GFC samples as HPLC didn't give me desired results.

I would open this to discussion in this research forum. Please go ahead.

Thanks