Exalpha Biologicals, Inc.

Product Highlight

FIX&PERM Cell Fixation and Permeabilization Kit

Flow cytometric analyses with monoclonal antibodies were so far mainly restricted to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such studies. Also excluded from flow cytometric studies were cytoplasmic localizations of well-established membrane molecules like CD3 and CD22, which, in their cytoplasmic form, are the most reliable lineage markers in undifferentiated leukemia. With the FIX&PERM® Kit flow cytometric analysis of intracellular antigens has become as easy as surface antigen studies. The only prerequisite is the availability of suitable antibody conjugates. Most of the available monoclonal antibody conjugates can be used with the FIX&PERM® Kit, some determinants are sensitive, however, to the fixation step involved. This and the optimal fixation time have to be tested for each reagent.

News

Welcome, Peter Rutten

We are pleased to announce that Peter Rutten has now started in his role as Operations Director here at Exalpha Biologicals Inc. Peter has a Master’s of Science degree in Industrial and Organizational Psychology and this has lead him down a very business orientated career path. Peter is looking forward to working with the team at Exalpha. Peters primary focus is the customer experience and he will be working with the Laboratory team, the Quality Control team and the Order Processing team to ensure this focus is achieved. We all wish Peter well in his new role.

Western Blotting Protocol

Lysate preparation

Lysate preparation will depend of the source of cells (Monolayer, Cell suspension or Tissue samples). In general, Cells are washed with cold 1xPBS, subsequently lysed with an appropriate buffer (i.e. RIPA buffer) containing freshly added protease inhibitors. Passed through a 21G needle to shear the DNA and then centrifuged at 10K rpm for 10min at 4°C. discarding the pellet. Determining the Protein concentration by BCA assay from Pierce.

Mix samples 1:1 ratio with 2x Sample loading buffer (Sigma S-3401) (10-60ug whole cell Lysate, 10-20µg nuclear extract, or 10-20ng purified protein per lane), and then boil the sample for 4 min. Unused samples can be stored at -20°C for up to several months.

Protein Separation and Detection

Separate protein samples and molecular weight markers by polyacrylamide gel electrophoresis.

Transfer protein samples from polyacrylamide gel onto nitrocellulose membrane using an electroblotting apparatus according to manufacturer’s protocols.

Block the nitrocellulose membrane for at least one hour at room temperature or overnight at 4°C on a rocking platform with TBST 5% non-fat dry milk solution using about 1 mL per cm2 of membrane.

Wash the nitrocellulose membrane 5 times in TBST for 10 minutes each on a rocking platform.

Incubate the nitrocellulose membrane for 60 minutes on a rocking platform with primary antibody diluted in TBST, 5% milk.

Wash the nitrocellulose membrane 5 times in TBST for 10 minutes each on a rocking platform.

Visualization

Incubate with second antibody enzyme conjugate diluted in 5% non-fat dry milk TBST for 60 minutes on a rocking platform. (Please see secondary antibody manufacturer’s recommendations for appropriate dilution)

Wash the nitrocellulose membrane 5 times in TBST for 10 minutes each on a rocking platform.

From our experience when using secondary Antibody conjugated to HRP, we recommend the use of SuperSignal West Femto Maximum Sensitivity Substrate from Pierce, when faced with low expression of proteins in the Lysate.

A beginning exposure time of 30 seconds is recommended. This should be adjusted either longer or shorter depending on results obtained.