Odyssey infrared imaging system

Direct infrared fluorescence detection on the Odyssey Infrared Imaging System provides the established standard for Western blot analysis that can’t be equaled with chemiluminescence and visible fluorescence. Infrared detection gives you the quantitative analysis and wide linear dynamic range that chemiluminescence cannot. Strong and weak bands on the same blot are accurately detected without the […]

Protocol for Poly-D-Lysine Preparation and Plate Coating

1. Prepare Poly-D-Lysine a. Add 100 ml of sterile tissue culture grade H2O to 5 mg poly-D-lysine (Sigma P6407). Final concentration of poly-D-lysine is 50 ug/ml. b. Mix by pipetting several times. c. Store at 2-8 ºC or –20 ºC. 2. Prepare Coated Plates a. Add enough poly-D-lysine solution to cover culture plate surface. b. […]

Easy (all purpose) brain protein extraction

If you just want a positive control for your western blot and you need rat or mouse brain, there’s no need for a fancy and long brain protein extraction.   – Dissect the animal and extract the brain –  Take the whole brain or cut a piece of it (the region you are interested in) and […]

Don’t throw the membranes away!

Did you know that you can let western nitrocellulose and PVDF membranes dry up and keep them like this for a long time? Yes, you can I was doing that in my lab in Spain and when I came to US, I realized a lot of people was not doing it. When you are done […]

Everything about western blots

Everything you always wanted to learn about western blots but were too afraid to ask is in this web site: http://www.westernblotting.org/ Western blot protocols, troubleshoting, definitions, buffers and theory all in a web site. What is biotin? how the stripping buffer works? how do I prepare the lysis buffer? In a very basic way, you’ll find your answers in this […]

Western blot troubleshooting

Here some problems we all have had when doing western blots: – high background signal – strange/non specific bands on the blot – low sensitivity – is a right band detected? – uneven results with lots of spots In the protocols section of the company Agrisera there is a good troubleshooting section that will answer […]

Western blot protocol

A complete western blot protocol from Howel lab in University of California San Diego. Here is the link: http://cancer.ucsd.edu/howelllab/Western.html From how to obtain a cell lysate to how to prepare the gels, running the gels, membrane transfer, detection and stripping the membrane. Also all the reagents and buffers you need for your western blot. There is […]

Cell trypsinisation

1. Remove medium from culture dish. 2. Gently wash cells in a PBS without Ca or Mg. 3. Remove the wash solution. 4. Add trypsin-EDTA solution to cover the bottom of the culture dish (2ml for 10cm dish, 0.5ml for 60mm dish). 5. Put dish in the 37°C incubator for up to 5 minutes. 6. […]

Heat inactivating serum

1. Place bottles in a 56oC water bath. Make sure the bottles cannot tip over or otherwise become submerged in the water bath. The temperature is critical for complement degredation, so make sure! 2. Wait until the temperature stabilizes at 56oC (after adding the serum to the water bath) 3. Gently swirl the bottles every […]

The basics about cell culture methods

The Department of Biological sciences of the University of Maryland (UMBC) has a protocol section very useful for cell culture first time users. They explain extensively all the components of the work area and the equipment mainly used, from how the hood works to the microscope or the incubators. Also, types of cells that grow […]