Immobilon® Membranes, Sandwiches and Blotting Filter Paper
Immobilon® PVDF membranes are the ideal transfer membranes for protein blotting applications.
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Overview
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Documentation
References
| Reference overview | Application |
|---|---|
| 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 LCGC 2010 Show Abstract 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 | Immunoblotting (Western) |
| 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 | Western Blotting |
| 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 | Western Blotting |
| 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 |
FAQ
| Question | Answer |
|---|---|
| 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. |
| I have been using Nitrocellulose for my Western Blots but am thinking of converting over to Immobilon-P PVDF membrane. Are there any differences in the protocol that I should know about? | Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes. Another change to note is that the SDS tolerances are not equivalent for the two membranes. The binding of protein to Immobilon P is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05%. |
| 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. |
| How should I store the Immobilon-P membrane? | Prior to use the Immobilon-P membrane should be stored at room temperature. After use the membrane may be stored by allowing the membrane to dry, wrap in plastic wrap and store at 4º C. For short term (overnight) storage the membrane may be stored wet by wrapping in plastic and refrigerating. |
| What's "blow-through", and how can I prevent it? | Blow-through is a phenomenon seen in blotting applications where proteins move from the gel and through the membrane without binding to it. This is usually seen with low molecular weight proteins and can be minimized by gradually increasing the current or voltage during the transfer and optimizing the composition of the transfer buffer. Also, add a back-up sheet of membrane to check for any proteins that still may get through. It should be noted however, that for N-terminal sequencing applications, Immobilon-PSQ is the membrane of choice. The increased surface area and tighter pore size of this membrane result in better retention of small molecular proteins than Immobilon-P. |
| I've completed my transfer to Immobilon-P, and now I see translucent areas on the membrane that correspond to the location of my wells or bands. Is there something wrong? | No. When protein is transferred to Immobilon-P, the membrane becomes hydrophilic where the protein is bound. If the membrane is dried, or even partially dried, and then wetted with an aqueous buffer, the regions with bound protein will appear translucent. |
| 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. |
| Can I use Immobilon P for nucleic acids? | Although, historically, there have been references that cited using Immobilon P with nucleic acids, Millipore strongly recommends that nucleic acids be transferred to either nitrocellulose or nylon membranes. Immobilon P is best suited for the transfer of proteins and there is an extensive body of literature within Millipore to support this application. |
| Can I use Immobilon-P for dot or slot blotting of my proteins? | Yes. If you are using a dot or slot blot manifold, first wet the Immobilon-P with 100% methanol, then rinse it with water, and then equilibrate in PBS or similar buffer. Place the Immobilon-P in the manifold, and add all samples before turning on the vacuum. If your manifold requires that one or more pieces of filter paper be placed under the transfer membrane, the filter paper that touches the membrane should also be wet with the buffer used to equilibrate the Immobilon-P. If you are not using a manifold, the Immobilon-P should be wet with methanol, rinsed with water, and equilibrated as above, and then placed on a piece of coarse filter paper (such as Whatman 3MM) that has been wet with the same buffer. It is helpful to place some dry paper, either Whatman 3MM or paper towels, beneath the wet Whatman in order to help reduce the tendency of the sample to spread laterally on the wet Immobilon-P. Following the spotting procedure (either with a manifold or without), for best results the membrane should be allowed to dry, then rewet with methanol and rinsed with water prior to blocking and immunodetection. |
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