I think that the best method is an HPLC-protein assay (please see files).   This method is c.a. 200-fold higher sensitivity than photometric assay, and analysis time is within 4 min per sample.   This method is applicable to hydrophobic membrane proteins and also to cell lysates.

Thank you so much Kou for nice suggestion, since our lab is concerned with biological assays for cancer, any other alternative technique like any kit or reagent based analysis would be easier and feasible for us. Is there any such kind of alternatives available for more accurate quantification of protein in samples for western blotting?

I think that clinical laboratory should have HPLC technology, since the cost for HPLC becomes to similar cost to non-quantitative ELISA, PCR, MALDI-TOF-MS, Western blotting (SDS-PAGE), radio-labelling, etc.    I only pray that you can obtain suitable financial support or good co-worker of HPLC technology.   

Thanks @Kou Hayakawa for your response. We do have HPLC and other sophisticated analytical facilities in our Institution. But I wonder how the cost of HPLC (Instrumentation, solvent and other) is less than Western blotting (SDS-PAGE). However, I just wanted to know alternatives for quantification of protein which is required for Western blotting (Signalling pathway elucidation in cancer biology).

Thanks...

 "how the cost of HPLC (Instrumentation, solvent and other) is less than Western blotting (SDS-PAGE). "   I have invented the HPLC-Soap-SEC, which is a high-recovery protein-separation method.   Therefore, separations can be achievable repeatedly or many times by using a single SEC column (please see file; .SEC column 300A silica).

Western blotting is not applicable to hydrophobic membrane proteins and Signalling pathway proteins.   Then, I would like to recommend the protein microseqencer or PDMD (protein-direct-microsequencing-deciphering) method to identify the hydrophobic component proteins (please see files; HepG2 fucoidan, JMBT Alopecia).

Thank you very much Kou Hayakawa  for all your suggestion!

As I have repeatedly declared, only HPLC-protein assay has the linear calibration line inserting to zero point.   If recovery of the protein from the column is adequate, RP-HPLC-protein assay is possible and reliable (please see file;  Lysozyme by RP-HPLC). 

All the photometric protein assays (such as Lowry's method, BCA (bicinchoninic acid) method, and Bradford's dye-binding method with Coomassie Brilliant Blue G-250) have bent calibration curves due to violation against the Lambert-Beer's law.  The Lambert-Beer's law says that purification is essential.

Non-photometric Kjeldahl method, which hydrolyze the sample by 36 N sulphuric acid at high temperature to measure the nitrogen content, requires non-general nitrogen‐protein conversion factor of 6.25 and long analysis time.

The method of acid hydrolysis followed by HPLC-amino acid analysis (please  see file; BIN pig brain) requires long hydrolysis time (6N HCl, 110 ℃, 22 h).   Both pre-column RP-HPLC-PTC-amino-acid analysis (1.5 h) and post-column HPLC-IEX-amino-acid analysis (2 h) using OPA are quantitative methods.  

If relative molecular mass (Mr) is known, PDMD method is also a precise protein determination method giving similar value to HPLC-Soap-SEC-protein assay (our unpublished observation).      

My sincere thanks to @Alaaeldin A. Hamza and @Kou Hayakawa for your valuable suggestion. The expert opinion by you guys is so helpful to me as well as other researchers. Thanks a lot !