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Description of the problem: in isotype control lane of the IP performed there is no typical strong IgG heavy chain band observed in the experiment lane Sample Nuclear extract digested with MNase Sample preparation incubation of sample with IP Ab’ ON 4 deg C, rotation 4 h with protein A dynal beads to form Matrix-antibody-antigen complex, extensive washes with RIPA buffer. Matrix used to allow precipitation of antibody-antigen complex invitrogen™, Dynabeads® protein A Detergent present in your system 0.7% DOC, 1% NP-40, 0.1% SDS Electrophoresis/Gel conditions SDS-PAGE 4-20% gradient gel Primary Antibody custom Mouse α hnRNP (all isoforms) Expected size, 35-40 kd. TBST 1% (sodime azide) BSA 4deg ON, 3 20 min wash with TBST Secondary Antibody Jackson Peroxidase-conjugated AffiniPure Goat α mouse IgG (H+L) (1:40000) in 5% skim milk, 3 20 min wash with TBST Detection method Autoradiography
Asked on Mar 08 2012
Thank you for contacting us and thank you for providing the protocol and image.
After reviewing the data presented I would like to suggest a few changes that may help improve your results.
Mouse IgG3 does not bind well to beads for IP. To overcome this we recommend that when using an IgG3 antibody that you incubate for a longer period and use higher concentration. As your primary antibody is IgG2b, which binds more strongly to protein A beads, a correct isotype control will be anti-Mouse IgG2b. We do offer this product, ab18421. I have placed the link below.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Mar 08 2012