Sumit:  We do both, but only ELISA is more quantitative for two reasons:  First, a standard protein has to be used for calculating the concentration of proteins in each well.  Second, enzyme reaction (HRP) takes place in each well thus the wells do not compete each other for substrate.  This is not the case for Western where each lane of proteins are exposed to HRP-antibody in the same tray, and heavier band may use more substrate than faint band.  However, ELISA has its own drawback: trace amount of serum proteins can not compete with abundant proteins in coating the plastic well, while dilution makes it too diluted for detection as well!  In that sense, Western blotting is a better choice for trace proteins.  Please see our paper for details (PMID: 25900303 or download from my account).  Good luck.

Could you provide more information (protein concentration in blood relative to total plasma or serum protein of animal? Do you have antibodies to use in ELISA or WB?

For most species, blood contains hundreds if not thousands of proteins in it.  The normal plasma protein for most species is around 5-7.5 grams/dL.  Unless you are looking at a protein present in very minor amounts (which I could not determine from your question) I see no reason to use ELISA.  Total protein my be quantitated using one of the many protein determination assays available form Pierce, BioRad or made up in lab. 

If you are looking at a minor blood protein, ELISA would be best.  If your ELISA is good, you may be able to detect nano-grams to picograms of protein present in serum/plasma.  However, you would need purified protein and an antibody to that protein to quantitate it.  Western blot would require nanograms to micrograms on most formats for gels. However, western blot would id only protein of interest? 

I'm looking for specific proteins concentration level in blood relative to serum protein of animal. WB seemed like a better choice than ELISA, and not sure if the proteins I'm gonna work with can be found in trace level (But antibodies are available). Also there's difficulties developing protocol for western blotting by determining blood serum as sample.

I would like to recommend HPLC-SEC protein determination for total protein of serum (please see file; prot deter summary).    Further, PDMD (protein-direct-microsequencing-deciphering) method is quantitative for component serum protein analysis, since serum has many hydrophobic membrane proteins (please see file; JMBT Alopecia). 

By the way, ELISA is not quantitative mainly due to violation of the Lambert-Beer's law.   Western blot is also non-quantitative method, since hydrophobic membrane proteins are not able to be electrophoresed and the addition of non-ionic detergent (such as 1% Triton X-100 and Nonidet P-40)  is necessary to fully migrate the hydrophobic proteins (our unpublished observation; please see file IEF for hydrophobic protein).

Dear Sumit

It will be interesting to precise kind of protein you need to determine and therefore we could  recommend you effecient method.

Regards

Marius

Dear Marius,

GPC3, CD34, and sFRP-4 these are the three protein I need to detect and quantity.

With my lab set up, I can't use either of HPLC-SEC or PDMD.

I suggest Picrate alkaline method for total protein and serum electrophoresis for the fractions

Sumit,

If you have antibodies, why not perform IP on some serum/plasma samples to pull down the proteins of interest (not quantitiative by itself but could provide you approximate range for serum/plasma concentrations).  I have used protein A/protein G coated magnetic beads to bind antibodies then add plasma/serum to beads in eppendorf.  Then place in magna rack or similar to pull down proteins.  We have placed serum/plasma + beads on rocker at 4C to mix, then pull down next day (may need to optimize binding and prevent proteolysis).  We also use low pH to dissociate protein from beads (may also want to look at Pierce for cross-linker to prevent antibody from eluting as well (simplify gel or ELISA).  Could be run on SDS-PAGE for WB or sequencing. 

Another comment - Is it possible to obtain recombinant proteins of interest?  Then ELISA can be quantitative?  This my help optimize IP protocol for each serum/plasma protein. 

Another thought - If you know your proteins of interest are not bound to other serum proteins, deplete albumin from sample prior to IP? 

Jeff

Thank you, everyone. 

The choice between ELISA and IB really depends on the number of samples, reagents availability, specificity and sensitivity you need. 

Thank you

I have decided to do ELISA

Dear Sumit

Process of ELISA is also expensive and time-consuming. ELISA method is recommended  for measuring protein in human serum or  plasma. It has high sensitivity. And yet some researches showed that quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM).

Find an example to determine protein concentration in Treseder Lab Protocol . Check this link: https://webfiles.uci.edu/treseder/public/Protocols/Calculation%20Examples%20for%20Bradford%20and%20ELISA.pdf

Please I recommend to read also this interesting publication entitled: Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma. J Proteome Res. 2013 Dec 6; 12(12): 5996–6003. doi: 10.1021/pr400877e. Direct link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864264/

Hoping to be helpful

Marius

I have recently found that IgD is a relatively hydrophobic protein, due thanks to Dr. Rajanya Banerjee (National Institute of Technology Rourkela, Sundargarh, Orissa, India).  

I have answered to her that "I must say something about the IgD purification, although I have never purified it yet. 

Immunoglobulin D (IgD) of humans is relatively hydrophobic protein (hydrophobicity of 0.522), which is found only in human breast milk (7.0 μg/mg of milk protein) among my biological specimens.   Hydrophobicity of immnogloblins are calculated using constant regions.   Human serum biotinidase has hydrophobicity of 0.535, and the purification with addition of non-ionic detergent is necessary (see file; SEC BIN).   Pig brain biotinidase has even lower hydrophobicity of 0.504, however the purification with addition of non-ionic detergent is necessary (see file; brain-BIN pig).   Membrane IgD of Ctenopharyngodon idella (grass carp) has hydrophobicity of 0.489, and may be easier to purify than human IgD.

Even weakly hydrophobic-glycoprotein of purified Ovotransferrin/Conalbumin (hydrophobicity; 0.518, an product of GE Healthcare) contains two hydrophobic proteins of Cadherin-related family member 1/Chicken photoreceptor cadherin (700-865; hydrophobicity 0.526), and Tubulin-specific chaperone D/Chicken tubulin-folding cofactor D (309-1019; hydrophobicity 0.571) (analyzed by microsequencing method; our unpublished observation). 

Hydrophobic glycoprotein of commercial Lactoferrin (Hydrophobicity 0.542; from American bovine milk; Sigma, L-9507; purity higher than 85%) also has many membrane glycoproteins (please see file; LF Dr. Kawakami (in Japanese)).

Thus, I would like to recommend the final-step purification of fish IgD by HPLC-SEC method containing non-ionic detergent".

Then, the notorious ELISA uses apparently pure IgG (hydrophobicity 0.505; with similar hydrophobicities to milk-type human biotinidase 0.505, brain-type pig biotinidase 0.504, kidney-type human biotinidase 0.573, kidney-type guinea pig biotinidase 0.515, and human fetal/Inflammatory-type biotinidase (reported by Dr. Barry Wolf et al.) 0.580), that still may have contaminated with hydrophobic membrane proteins (serum has been found to contain many hydrophobic membrane proteins; see file JMBT alopecia).    

Therefore, I would like to recommend again to use quantitative HPLC method instead of notorious ELISA method.   I would like to pray that your country shall choose and support financially the reliable HPLC method.