|Mobile Phase Preparation for UHPLC: Membrane Filtration Method Affects System Performance and Leaching of Extractable Impurities|
Subodh Kulkarn(1), Jesmi George(2) and Vivek Joshi(2) (1) Millipore India Pvt. Ltd., Bioscience Division, 50A, 2nd Phase, Ring Road, Peenya, Bangalore, India 560058 (2) Millipore Corp., Bioscience Division, 17 Cherry Hill Drive, Danvers, MA 01923
UHPLC/UPLC® is a revolutionary chromatography technique that is gaining wide acceptance among researchers due to improved resolution, shorter chromatographic runs, and the capability for doing fast method development. The presence of sub-2 µm particles in UHPLC columns provide these benefits but also poses challenges in sample and mobile phase preparation. Particulate impurities in the sample or mobile phase can cause backpressure buildup in the UHPLC system, causing system failure. In fact, most UHPLC instrument vendors recommend filtration of mobile phase using 0.2 µm filters, but there is a lack of data showing the benefits of filtration. In this article we describe the filtration of mobile phases through syringe filters of varying pore size and membrane type, followed by analysis by UHPLC and mass spectrometry. Our results clearly indicate that filtration of mobile phase components using the optimal membrane filter will help protect UHPLC systems from particulate impurities that may clog and shut down the system, increase the sensitivity of detection, and improve the accuracy of quantitation.Full Text Article
|Quantitation of Protein on gels and blots by infrared fluorescence of Coomassie blue and fast green|
Luo S., Wehr N.B., Levine R.L.
Analytical Biochemistry:350 (2006):233-238 2006
|Pre-B-cell colony-enhancing factor is a secreted cytokine-like protein from the human amniotic epithelium.|
Ognjanovic S, Ku TL, Bryant-Greenwood GD.
Am J Obstet Gynecol. 2005 Jul;193(1):273-82 2005
|Role of the Small Heat Shock Proteins in Regulating Vascular Smooth Muscle Tone|
McLemore E.C., Tessier D.J., Thresher J., Komalavilas P., Brophy C.M
J. Am. Coll. Surg. 2005, Vol 201 (1):30-36 2005
|A high-affinity reversible protein stain for Western blots|
Antharavally B.S., Carter, B., Bell, P.A., Mallia K.
Analytical Biochemistry 2004,Vol 329:276-280 2004
|Biochemical analysis of GABA receptor subunits alpha 1, alpha 5, beta1 beta2 in the hippocampus of patients with Alzheimer's disease neurophathology|
Rissman, R.A., Mishizen-Eberz A.J. N.,Wolfe, C.B.B., DeBlas A.L., Miralles C.P., Ikonomovic M.D., armstrong D.M.
Neuroscience 120 (2003) 295-705 2003
|Proteomics reveals protein profile changes in doxorubicin treated MCF7 human breast cancer cells|
Cheng S.T., Pan T, L., Tsai Y.Ch, Huang C. M.
Cancer letters 2002. vol 181:95-107 2002
|Towards proteome-wide production of monoclonal antibody by phage display.|
Bin Liu, Lan Huang, Carina Sihlbom, Al Burlingame and James D. Marks.
J Mol Biol. 2002 Feb 1;315(5):1063-73 2002
|Mass Spectrometry Sample Prep|
|Characterization of retinoic acid receptor-deficient keratinocytes.|
Goyette Philippe; Chen Chang Feng; Wang Wei; Seguin Francois; Lohnes David(a)
Journal of Biological Chemistry v 275 pg 16497-16505 June 2, 2000 2000
|Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation|
Dmitrieva Natalia(a); Kultz Dietmar; Michea Luis; Ferraris Joan; Burg Maurice ; Journal of Biological Chemistry v 275, pg 18243-18247 June 16, 2000
Journal of Biological Chemistry v 275, pg 18243-18247 June 16, 2000 2000
|Should I prewet MultiScreen Immobilon-P plates before use?||Yes. Use 15 ul of 70% ethanol or methanol to prewet the Immobilon P membrane. This membrane is hydrophobic and requires a prewet to allow liquid to pass through the membrane.|
|What is the thickness of the Immobilon PSQ?||Immobilon PSQ is 200 microns thick.|
|What is the smallest size of a protein or peptide which binds to the Immobilon-PSQ?||For the PSQ we do not suggest that they go lower than 2 kDa for the protein size. To help promote the transfer of the smaller proteins the methanol concentration of the transfer buffer can be increased to 20% (w/v) and the SDS lowered to 0.01%. The strength of the electric field can also be reduce by 50% to increase the proteins contact time with the membrane. Standard stains can be used such as Coomasie Blue and Ponceau. Keep in mind that some of the stain such as Coomasie Blue are not reversible.|
|How many times can I strip and reprobe Immobilon-P?||While 2-3 times is probably the limit, it is difficult to give an absolute number in regards to stripping and reprobing. This is because there are many factors to consider when it comes to protein binding to PVDF and primary antibody affinity to these proteins. Different proteins will have varying degrees of affinity to PVDF. This is based on factors discussed in our Protein Blotting Handbook (see TP001; Protein Binding section). One round of stripping may remove one protein and leave another intact. The same could occur when using different primary antibodies. Different antibodies will have varying affinities to different proteins. If carrying out several rounds of stripping and reprobing, one strategy might be to detect the least abundant proteins earlier leaving the higher abundant proteins later for detection.|
|What is the difference between Immobilon–FL and Immobilon-P?||Both membranes are made from PVDF. The difference in background fluorescence is due to proprietary modifications in the membrane manufacturing process.|
|What fluorescence-based detection methods can be used with Immobilon-FL?||Immobilon-FL can be used in Western blotting applications using either fluorescent dye-conjugated antibodies or chemi-fluorescence substrates. Western blots can be imaged in the visible or IR range. Single color detection or multiplexing for co-localization studies can be performed efficiently on Immobilon-FL Fluorescent proteins, e.g. green fluorescent protein, (GFP) or proteins tagged with GFP, blotted onto the membrane can be readily detected as well.|
|What fluorescent dyes are recommended to use with Immobilon-FL?||Immobilon-FL membrane exhibits very low background fluorescence over a broad range of wavelengths and can be used with all visible and infrared fluorescent dyes. Generally, dyes with larger Stokes shift (greater separation between absorption and emission wavelengths) are preferred in western blotting applications using fluorescence-based detection. NOTE: To prevent photo-bleaching, protect the membrane from light during secondary antibody incubations and washes until the membrane is ready to be scanned.|
|How long does the fluorescent signal stay on the developed blot?||The fluorescent signal from fluorescence-tagged antibodies will remain stable on the membrane for several months, or longer, when stored protected from light. The membranes may be stored dry or in PBS at 4ºC. The fluorescent signal from chemi-fluorescent substrates does not remain for long so blots using these substrates need to be scanned immediately.|
|Do I need to dry Immobilon-FL blots before scanning?||Drying the blot may enhance signal strength. NOTE: Do not wrap the blot in plastic/saran wrap while scanning. If you do need to wrap the membrane, Mylar should be used.|
|Can Immobilon-FL be stripped and re-probed?||Yes, Immobilon-FL can be stripped and re-probed like any other PVDF membrane, using standard detergent or acidic methods (refer to Millipore’s Protein Blotting Handbook). Keep the blot wet. If dried, rewet with methanol and then rehydrate before stripping the blot. Once the membrane has dried the stripping is ineffective. Alternatively, the blot can be photo-bleached to remove the fluorescence.|