37. Cleaved
You can incubate 20ul of bed volume saturated resin with 0.1, 0.5, 1 & 2 Units of thrombin (in 20ul total PBS) 2hr, 4hr, 6hr & ON at 22C (or RT). Stop reaction by spinning for 4min 3000rpm, separate supernatant from beads, and add PAGE-SDS sample buffer + 1mM PMSF to the supernatant, and keep immediately at -20C until use. Analyze PAGE-SDS gels versus a control of non-cleavaged protein (eluted non cleaved protein). Longer incubation, more enzyme or higher temperature will increase cleavage; while lower incubation, less enzyme or lower temperature will decrease cleavage.
37. Cleaved
1. For 1ml of bed volume saturated resin, mix 50Units of thrombin solution in 1ml PBS. Gently swirl to mix. Shake or rotate at 22C (or RT) 2-16hrs 2. Spin the suspension 4min 3000rpm to pellet the beads. Keep supernatant aside. You can stop protease cleavage with 1mM PMSF or AEBSF (more stable). 3. Extract beads twice or more with 1ml PBS or buffer. Keep each supernatant aside. 4. As a control you can elute the remaining uncleaved protein (still attached to the resin through the GST) by extraction several times with elution buffer. 5. Check results by PAGE-SDS gels or immediately separate the cleavageproducts by chromatography. 6. When a satisfactory condition is found, scale-up the reaction proportionally.
BACKGROUND: The cleavage of recombination signals (RS) at the boundaries of immunoglobulin V, D, and J gene segments initiates the somatic generation of the antigen receptor genes expressed by B lymphocytes. RS contain a conserved heptamer and nonamer motif separated by non-conserved spacers of 12 or 23 nucleotides. Under physiologic conditions, V(D)J recombination follows the "12/23 rule" to assemble functional antigen-receptor genes, i.e., cleavage and recombination occur only between RS with dissimilar spacer types. Functional, cryptic RS (cRS) have been identified in VH gene segments; these VH cRS were hypothesized to facilitate self-tolerance by mediating VH --> VHDJH replacements. At the Igkappa locus, however, secondary, de novo rearrangements can delete autoreactive VkappaJkappa joins. Thus, under the hypothesis that V-embedded cRS are conserved to facilitate self-tolerance by mediating V-replacement rearrangements, there would be little selection for Vkappa cRS. Recent studies have demonstrated that VH cRS cleavage is only modestly more efficient than V(D)J recombination in violation of the 12/23 rule and first occurs in pro-B cells unable to interact with exogenous antigens. These results are inconsistent with a model of cRS cleavage during autoreactivity-induced VH gene replacement. RESULTS: To test the hypothesis that cRS are absent from Vkappa gene segments, a corollary of the hypothesis that the need for tolerizing VH replacements is responsible for the selection pressure to maintain VH cRS, we searched for cRS in mouse Vkappa gene segments using a statistical model of RS. Scans of 135 mouse Vkappa gene segments revealed highly conserved cRS that were shown to be cleaved in the 103/BCL2 cell line and mouse bone marrow B cells. Analogous to results for VH cRS, we find that Vkappa cRS are conserved at multiple locations in Vkappa gene segments and are cleaved in pre-B cells. CONCLUSION: Our results, together with those for VH cRS, support a model of cRS cleavage in which cleavage is independent of BCR-specificity. Our results are inconsistent with the hypothesis that cRS are conserved solely to support receptor editing. The extent to which these sequences are conserved, and their pattern of conservation, suggest that they may serve an as yet unidentified purpose. 041b061a72