|Western Blotting Tools|
|Protein Blotting Handbook: 6th Edition|
|Protein Dot Blotting using Immobilon-P|
|Immobilon Transfer Membranes: For superior protein and nucleic acid blots|
|History of Western Blotting|
|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
|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|
|Amino Acid Analysis: A comparative Study of stains on PVDF membranes|
,Journal of Biomolecular Techniques, Vol II, Issue 1, March 2000.
Journal of Biomolecular Techniques, Vol II, Issue 1, March 2000. 2000
|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
|Can I use the Rapid Immunodetection method with nitrocellulose?||No, the surfactants used in the manufacture of nitrocellulose allow the membrane to wet out in all aqueous buffers. Immobilon-P is the membrane of choice for this method.|
|What is the Rapid Immunodetection Method?||Rapid Imunodetection is a method used with Immobilon-P that eliminates the blocking step by exploiting the inherently hydrophobic nature of the membrane. This hydrophobicity excludes the aqueous antibody solution from the internal pore structure of the membrane, thereby eliminating the need for a blocking step and greatly reducing the amount of washing required to remove excess immunoreagents.|
|How can I transfer a range of high and low molecular proteins from my gel onto Immobilon-P without the small proteins being lost?||Ramping of the current and optimization of the transfer buffer composition can be used together to give efficient blotting of proteins over a wide range of molecular weight. They may also be used separately to give the maximum transfer efficiency for either small or large proteins.
1. Ramp - To help slow down small proteins, start the transfer at half the normal current (or voltage), and gradually increase the current (or voltage) during the transfer period to the maximum setting recommended. This measure allows the smaller proteins to travel more slowly through the electric field and have more residence time within the membrane. This step increases the chance that binding will occur. As the current is increased the larger proteins start to move out of the gel and onto the membrane as well.
2. Optimize your transfer buffer - When optimizing your transfer buffer, it is important to realize that conditions favoring the transfer and binding of small proteins often have a negative effect on the transfer of large proteins. The converse is also true. The following rules are generally applicable. Most small proteins elute from a gel with little difficulty. To improve their binding to the membrane, SDS should be omitted from the transfer buffer and the methanol concentration should be increased to as high as 40%. For optimal blotting of large proteins, decreasing the methanol concentration to 10% or less and supplementing the transfer buffer with up to 0.05% SDS enhance their solubility and minimize their precipitation within the gel. Thus, they are better able to elute from the gel. Large proteins normally bind to the membrane very readily.
|I used the rapid immunodetection method with Immobilon-P and noticed higher backgrounds. What happened?||The membrane wasn't completely dry. Try dipping the membrane in 100% Methanol after transfer and allow to dry in a hood for 15 minutes prior to exposure to the primary antibody.|
|How can I make Immobilon-P transparent?||Immobilon-P can be rendered transparent for use in densitometry studies, by immersion in a 3:2 solution of DMSO:ethanol. If the membrane has been allowed to dry it is necessary to prewet the membrane with methanol before starting this procedure.|
|I transferred my protein to Immobilon-P and I can't find it. I've stained the gel and the membrane and nothing is there. What happened?||If the pI of your proteins are greater than the pH of the transfer buffer, the proteins will travel in the opposite direction. The protein probably transferred into the running buffer. If you suspect that your protein has a high pI, try CAPS buffer and/or put membrane on both sides of the gel.|
|How can I elute protein off Immobilon-P?||Several methods have been used depending on the nature of the study. Proteins can be eluted from the membrane by incubation at room temp. in 50 mMTris-HCL, pH 9, containing 2% SDS and 1% Triton X-100, followed by dialysis to remove most of the detergents. If the eluted protein is to be purified by HPLC, hydrogenated Triton-X-100 should be used as it will not contribute any UV absorbence to the chromatogram. If detergents in the protein solution are a problem, a 40% acetonitrile solution in 0.1 M ammonium acetate, pH 8.9 for 3 hours at 37oC can be used to elute the proteins. Lyophilization of the extract will remove the volatile solvents. If the eluted protein is to be purified by HPLC, hydrogenated Triton-X-100 should be used as it will not contribute any UV absorbence to the chromatogram.|
|What is the sensitivity of the transillumination method with Immobilon-P and how does it work?||The sensitivity of transillumination is equivalent to Coomassie Blue staining. After drying the membrane, the PVDF is re-wet in 20% methanol and viewed in white light, or placed in a tray containing 20% methanol on a white light transillumination box.
This method takes advantage of the change in refractive index of the rehydrated protein band to that of the water/methanol solution. The naturally hydrophobic Immobilon P membrane will not re-wet and, therefore can be visualized by the difference of refractive indices. The protein pattern appears translucent against the white membrane.
|Can Ponceau-S be used to stain proteins transfered to Immobilon-P?||Ponceau-S can be used with Immobilon-P and is reversible. The stain produces pinkish bands on a light background. The stain can be removed by washing the blot with 0.1N NaOH.|
|What are the important factors to consider when choosing a stain for use with Immobilon-P?||The most important factor in choosing a stain is how the proteins on the blot are to be analyzed. For example, if immunodetection analysis will follow, non-reversible stains are not recommended. Non-reversible stains generally exhibit the best sensitivity but can interfere with or prevent further analysis of the proteins. Although less sensitive, reversible stains allow assessment fo the blot and then can be washed from the membrane. However, there is a risk that some proteins may undergo chemical modification during the process or be washed from the membrane.|
|Immobilon -P Blotting Sandwiches|
|Immobilon®-E Transfer Membrane User Guide|
|Re-Blot Plus Western Blot Recycling Kit|