You will have to use light scattering along with size exclusion. Do you mean you want to use pre-stained SDS gel ladder for this? If ladder proteins are similar in shape then they will elute in designated order, and I am preety certain that they are not. Those gel ladders are not recommended as a reference tool for perfect calibration. You may use calibration standard mix provided by GE

Dear Mahesh,

Thank you very much for your prompt response. I will take note of your points on the pre-mixed markers.

The calibration marker kits including the one supplied by GE were designed for high resolution gel media and not for HPLC columns (made of silica, C18). I wonder if I can run the proteins onto smaller HPLC columns (reverse phase). The proteins that I wish to investigate in my study were supplied in a very low amount. It is difficult to run the proteins on bigger columns for precise elution and detection. Hence, I am thinking to use HPLC as a platform for standardization and to estimate stokes radius of the protein of interest.

I would appreciate if you could kindly advice me further.

Thank you.

Regards,

Bavani

I googled this- "how to separate proteins by RP-HPLC". Many answers. Here is one.

PROTEIN | Determination and Characterization

D. Otter, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

Reversed-phase chromatography

Separation of proteins by reversed-phase chromatography (RPC) depends on the reversible adsorption/desorption of the proteins, which have varying degrees of hydrophobicity, to a hydrophobic stationary matrix.

RPC has only recently become the method of choice for the separation and characterization of proteins/peptides. This is because of concerns that the organic solvents, e.g., acetonitrile, routinely used for the mobile phase can lead to protein denaturation and loss of biological activity. Strategies such as using short columns of short alkyl chain length, e.g., C4, organic modifiers such as ethanol or isopropanol, and omitting trifluoroacetic acid have been developed to allay these apprehensions.

Read full chapter

Yes of course you can use a mixture of proteins to create a calibration curve with your SEC HPLC method. We do this all of the time as a matter of routine. A typical HPLC UV detector is needed for a simple analysis, though a refractive index detector working under isocratic conditions only could also be used (no need for a fancy LALLS detection system unless you really want to make specific measurements).

You can purchase protein standards which are appropriate for your own work and mix up your own solution, then inject one sample containing the mixture of standards. As noted, some companies also sell pre-made solutions too, but often you are better off buying your own standards (the ones you want and need for your calibration), rather than use a mixture someone else prepared. You can also control the actual concentrations used, "freshness" and composition better if you prepare the mixture from individual standards. Please use at least five (n= > 5) stds to create your calibration table.

Please keep in mind that any "calibration" curve derived from a typical protein mixture may not provide accurate Mw values for your own samples. This is because your actual sample may not be in the same configuration (Conformation) as the protein standards you selected (think about each ones structure, in solution, and their hydrodynamic volume and size).

Yes. However, the retention mechanism or mechanism that separates your proteins may not be based on the stokes radius or molecular weight. Thus, you may have peaks that elute in a different order. Do you have a another means of identifying these peaks (proteins)? Thus, many separation scientists use individual 'pure' reference standards.

Dear Dr. David Arthur Carlson

Thank you very much for the input. I will go through the full chapter soon.

Dear Dr William Letter

Thank you very much for the information. It certainly solved my doubts. Actually, I found very few articles or sources that reported use of HPLC for standardization. That made me to wonder if there will be any complication to run it such as clogging and etc.

I will take note of your points on the sample prep and analysis. Shall try to run the samples soon.

Thank you once again.

Dear Dr Bruce Neagle

Thank you very much for your input too. I understand your point on the elution order may not reflect the actual properties of the proteins. As I mentioned in one of my response above, I will be handling two proteins which are supplied in very limited amount. After several considerations, I decided to try using HPLC to save the amount of protein used since I will be using smaller column size. I will be able to optimize the run several times, if needed conveniently for the standardization process too.

I am planning to determine and compare stokes radius of the native and modified versions of the protein using the standard curve that will be derived using the protein markers eluted on the same column. I understand that it may not reflect the actual values. However, it should be serve to make justifiable quantitative and qualitative comparison between both native and the modified proteins.

Thank you.

As stated earlier, you can setup a method to provide you with qualitative data using a basic HPLC setup. *Your choice of standards must be appropriate for your sample. More advanced detectors and standards would be needed to obtain accurate measurements, but for doing a comparison only, this may provide you with what you need at this point in time.

Sourav: You may not be familiar with HPLC SEC analysis of proteins using aqueous phases only (salts), which are commonly used in the HPLC field. Most biological compounds are analyzed in this way. No organic solvents are used to denature the proteins. No TFA either.

Their should be no worrisome interactions with the various protein standards in solution. Mixtures are used often for just this purpose.

Dear Dr. William,

Sure, I will be careful in the selection of the proteins. I hope it will work fine for the purpose of comparison.

Thank you.

Dear Dr. Sourav,

Thank you very much for your feedback.

Initially I had the same doubts. However, I managed to locate an article which performed standardization using reverse phase HPLC and water:acetonitrile gradient system.

I understand it is not quite common. I have been consulting my colleague regarding this matter too. I understand that I have a choice of running the samples using buffer as the mobile phase. I will plan out the experiment well before proceeding.

Thank you.

Regards,

Bavani

Dear Dr. William,

May I verify the use of water:acetonitrile as the mobile phase? I will not be adding TFA as it is not allowed at the place that I am going to use the facility.

Regards,

Bavani

It IS very common. SEC using a type of Ion-exchange chromatography. Make sure your proteins vary enough in size to be resolved first. Not everything can be separated and you have not provided any information regarding the proposed sizes you wish to compare so all information and suggestions presented so far are generalized. Take the time to learn some basic about proteins and denaturing before proceeding too (organic solvents generally denature proteins).

Your time would be better spent specifying the sizes you wish to compare, then researching examples in the literature (via the web and of course books!) on your own. Asking very generalized questions on forums like this one will likely lead to equally generalized answers. Learning HOW to research answers to questions is one of the most important skills you can learn and acquire. Having others simply provide you with detailed answers will not provide you with an opportunity to learn.

Dear Dr. William,

I wanted to try to use HPLC for the separation of proteins and standardization purpose due to limitation in the amount of the unknown samples. I am in the midst of exploring and learning the technique. As I have posted earlier, I wanted to get expert opinions on the possibility to use pre-mixed markers on the column. It is a specific technical question to see if it is feasible rather than running individual proteins.

I have list of markers which I plan to use for the standardization process. Following are the proteins cytochrome c, myoglobin, alpha-chymotrypsinogen, carbonic anhydrase and BSA ranging from 12.3-66kDA. This range will cover the predicted molecular weight for the samples. I have performed standardization using conventional Sephadex/Sephacryl columns previously.

As for the solvent system, I am aware that I can use buffer as the mobile phase but to admit I wasn't sure on the use of organic solvents due to some concerns. It was just recently that I learnt it is possible based on reading. Since it was brought up here in our discussion too, I took the opportunity to seek your personal input and guidance.

I hope you will understand that I am in the process of reading and learning the system and protocol before I could execute the work. I used this platform for active learning too. If required, I would have provided more details.

I appreciate your input and will certainly take into consideration in my research design.

Thank you.

It sounds like you now have enough basic information to make an informed decision and try some things out. Be sure and do so under the guidance of someone local who has practical experience separating proteins by HPLC. This is a very easy HPLC application, but can be complicated by co-elution when the needed resolution is not achieved.

• A few minutes on the web, using keywords and search engine such as GOOGLE, will provide anyone interested with hundreds of examples of protein mixtures run in aqueous SEC mode HPLC for use as Mw markers. This is one of the most commonly performed general analysis types using HPLC and LC-MS techniques.

Dear Dr. William,

Sure noted. I will do the needful.

Thank you.

Hi, I found you question to be interesting. Could you please share the results after running SEC standard protein marker on Reverse Phase column and what were the return parameters? Regards,

Harleen

Hi Dr. Harleen,

Sure I will share the findings once I complete the part of work soon.

Regards,

Bavani