rev: Augist 12, 2004
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For IMMUNOPRECIPITATION WORK
RDI Divison of researchd Industries Intl offers a wide line of antibodies. In
many situations, you may need a labelled secondaty antibody for the particular
application. You can access below "SOME" of the various specialty antibodies
we can provide. In most situations, the antibodies probably found in your
lab will work fine, however, in special situations, you may need one of these
specialty reagents (especially when working with antibodies on various animal
tissues). We offer many different types of antibodies to help solve this
problem. Please inquire for other applications or types of antibodies not
Additional Immunoprecipitation Reagents
$344.00/vial $312.00/vial 3 or more
-The following agarose conjugated antibodies are designed for the rapid and efficient collection of antibodies and proteins, including recombinant fusion proteins, from a complex protein mixture. antibody affinity gels are useful for selection of immunoglobulins and immunoprecipitation of antigens from sera, antisera, ascitic fluid or culture fluid, bacterial and mammalian cell lysates and fusion protein preparations. All the following immobilized antibodies are IgG fractions of antisera coupled to cyanogen bromide activated cross-linked 4% agarose. The antibody is used to remove a minimum of 5mg of antigen. The conjugate is supplied as a 1:1 suspension in PBS with azide. FOR IN VITRO RESEARCH USE ONLY-NOT FOR USE IN DIAGNOSTICS.
|RDI-IPBOVabG||Goat anti-Bovine IgG:Agarose 20mg|
|RDI-IPCHKabG||Goat anti-chicken IgG:agarose 20mg|
|RDI-IPGTabR||Rabbit anti-Goat IgG:agarose 20mg|
|RDI-IPHUabG||Goat anti-human IgG H+L:agarose 20mg|
|RDI-IPHUCabG||Goat anti-human IgG F(c):agarose 20mg|
|RDI-IPHUCabR||Rabbit anti-human IgG F(c):Agarose 20mg|
|RDI-IPRBabG||Goat anti-rabbit IgG:Agarose 20mg|
|RDI-IPMSabG||Goat anti-mouse IgG:Agarose 20mg|
|RDI-IPRTabG||Goat anti-Rat IgG:agarose 20mg|
|RDI-IPPRAabG||Goat anti-Protein A:Agarose 20mg|
|RDI-IPPRGabR||Rabbit anti-Protein G:agarose 20mg|
|RDI-IPGSTabG||Goat anti-GST:Agarose 20mg|
|RDI-IPBGALabR||Rabbit anti-B-Gal:Agarose 20mg|
|RDI-PRA-SEP1||Protein A:Sepharose 2ml $281.00|
|RDI-PRA-SEP2||Protein A:Sepharose 5ml $438.00|
|RDI-PRA-SEP3||Protein A:Sepharose 25ml $1250.00|
1) Prepare lysate by incubating 5 X 10 E07 cells in lysis buffer for 30 to 60 minutes
2) Vortex lysate and centrifuge for 10min at 250 X g to remove nuclei. Retain supernatant
3) Clarify supernate by centrifugation for 30min at 100,000 X G or microcentriguge for 30min at 10,000 X G
4) pretreat lysate to remove nonspecific protein binding by adding agarose conjugates. Use 10ul of control agarose per 200ul lysate, Shake for 1 hr at 4 DEG C. Centrifuge at 200 X g. Save supernatant.
5) Add 200ul of pretreated lysate containing antigen to each of two microfuge tubes. Bring volume to 1ml with dilution buffer.
6) Add primary antibody to one tube. For poyclonal antiserum or ascites fluid use 0.5 to 5.0ul. For tissue culture supernatant use 10 to 100ul. To the second tube add an equivalent volume of control for primary antibody. Incubate on ice for 1 hour.
7) For immunoprecipitation add 50ul of agarose conjugate per tube. Mix with gentle shaking for 1 hour at 4 DEG C
8) Centrifuge tube 1 min at 200 X g or microcentrifuge for 5 seconds. Carefully remove the supernate with a pipette. Gently resuspend pellet in 1ml dilution buffer. Repeat with a wash in TBS and then a final wash in 0.5M TRIS, pH6.8
9) Centrifuge again as above. Add 20ul to 50ul of sample buffer. Mix and heat for 5min at 100 DEG C. Microcentrifuge briefly and apply supernate directly to nonreducing SDS-PAGE. If reducing conditions are desired, transfer the supernate to a new tube and add 5% 2-mercaptoethanol. Mix and heat as above.
10) Electrophoresis protein mixture. Stain gel or immunoblot to visualize. Bands present will include polpeptides of antigen and antibodies used.
copyright by owner(s). RIPA= PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS (this may be made in large volumes. Add inhibitors at time of use from the following stock solutions).
a) 10mg/ml PMSF in isopropanol (use at 10ul/ml)
b) Aprotinin (Sigma cat#A6279, use at 30ul/ml)
c) 100nM sodium orthovanadate in frozen aliquots (use at 10ul/ml)
* Note:NP-40 is available through United States Biochemical/Amersham. A substitute may also be used=Igepal CA-630 (which is chemically indistinguishable from NP40 for most applications). This is available from Sigma Chemical.
Electrophoresis Buffer: 1.0ml glycerol; 0.5ml B-mercaptoethanol; 3.0ml of 10% SDS; 1.25ml 1.0M Tris-HCL pH 6.7; 1-2mg bromophenol blue. Store frozen in small aliquots. Alternatively make buffer without B-mercaptoethanol and store at room temp. Add B-mercaptoethanol just before using.
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