|Protein Dot Blotting using Immobilon-P|
|Immobilon Transfer Membranes: For superior protein and nucleic acid blots|
|Low Background Membrane for Fluorescent Protein Detection in Western Blotting|
|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
|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
|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
|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|
|The transcription factor EGR-1 directly transactivates the fibronectin gene and enhances attachment of human glioblastoma cell line U251|
Liu Chaoting(a); Yao Jin; Mercola Dan; Adamson Eileen
Journal of biological chemistry Vo- 275 Is- 27 Pg- 20315-20323 July 7, 2000 2000
|The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase.|
Ota Satoshi; Hazeki Kaoru; Rao Navin; Lupher Mark L Jr; Andoniou; Christopher E; Druker Brian; Band Hamid(a)
Journal of biological chemistry Vo- 275 Is- 1 Pg- 414-422 January 7, 2000 2000
|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.|
|How does the protein binding capacity and protein retention of Immobilon-FL compare to Immobilon-P?||Immobilon-FL’s protein binding capacity and protein retention are comparable to Immobilon-P.|
|Is Immobilon-FL better than Nitrocellulose membrane for Fluorescence detection?||Yes. Background fluorescence of Immobilon-FL is typically 2-5X lower than that of nitrocellulose, thus improving signal-to-noise ratio (sensitivity). Nitrocellulose membranes have other disadvantages. If allowed to dry out, they become brittle, tend to fracture and are difficult to handle. They are not recommended for stripping and re-probing. Nitrocellulose blots need to be scanned as soon as possible after detection, as diffusion of signal on a wet membrane may also occur.|
|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 is the level of detection sensitivity when using Immobilon-FL?||Immobilon-FL exhibits the lowest background fluorescence of any blotting membrane thus improving the signal-to-noise ratio (sensitivity) of any fluorescent dye. The fluorescence signal is dependent on the fluorescent dye used, thus the ultimate sensitivity of detection is determined by the dye itself. With Immobilon-FL the sensitivity is not limited by the membrane, e.g., detection of proteins in the low picogram level has been observed with QDot® Nanocrystals fluorescent-tagged antibodies on Immobilon-FL.|
|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.|
|What are the recommendations for two-color western / multiplex detection?||The two antibodies must be derived from different host species, so that they can be differentiated by secondary antibodies of different specificities. Before combining the two primary antibodies test their banding patterns on separate blots to know where to expect the bands. Use highly adsorbed cross-adsorbed secondary antibodies in two-color detection. Refer to the directions provided with the detection reagents for additional details.|
|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.|
|Are there specific recommendations on using 'low fluorescent’ reagents (buffers, blocking agents, etc.) with Immobilon-FL?||Immobilon-FL has been tested with the most commonly-used blocking reagents and buffers such as TBST, PBST, dry milk, BSA, casein and buffers recommended by the fluorescence scanner manufacturers, e.g., Licor Odyssey buffers. All were found to be compatible with fluorescent detection on Immobilon-FL. However, as in any western blotting experiment, optimization of blocking reagents and buffers may be required, depending on the nature of the antigen, antibodies and fluorescent dyes used. The use of freshly prepared buffers is always recommended.|
|Immobilon -P Blotting Sandwiches|
|Immobilon®-E Transfer Membrane User Guide|
|Immobilon®-P Transfer Membrane User Guide|
|Re-Blot Plus Western Blot Recycling Kit|