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Western blot analysis of Pax4 was performed by loading the indicated amounts of recombinant Pax3 (negative control) or Pax4 proteins, and 10ul of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. Pax4 was detected at ~38kD using a Pax4 polyclonal antibody (Product # PA1-108) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugate goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for at least 30 minutes. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: The presence of Pax3 recombinant protein was determined by probing a duplicate blot with a Pax3 polyclonal antibody (Product # PA1-107), and the corresponding image is on the webpage for Product # PA1-107.
|Tested species reactivity||Rabbit|
|Host / Isotype||Goat / IgG|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7.6, with 15mg/ml BSA, 50mM sucrose|
|Storage Conditions||4° C|
|Tested Applications||Dilution *|
|Immunohistochemistry (IHC)||1:5,000 - 1:100,000|
|Immunoprecipitation (IP)||1:500 - 1:5,000|
|Western Blot (WB)||1:5,000 - 1:200,000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Product # 31460 has been successfully used in Western blot, IF, ICC, IHC, IP and FACS applications.
Product # 31460 reacts with the heavy chains of rabbit IgG and with the light chains common to most rabbit immunoglobulins, but does not react against non-immunoglobulin serum proteins. However, this antibody may cross-react with immunoglobulins from other species and with SuperBlock® Blocking Buffers.
Store product at 4°C until opened. To extend the shelf-life of this product, add an equal volume of glycerol to make a final concentration of approximately 50% glycerol and store at -20°C.
Reconstitute with 2.0 ml of distilled water (0.8 mg/ml after restoration).
Thermo Scientific Anti-Rabbit secondary antibodies are affinity-purified antibodies with well-characterized specificity for rabbit immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.