Transcription, Translation of S35-Radiolabelled Protein and Binding to GST
Prepare the template by linearizing 25ug plasmid DNA at the 3'' end of the insert. Phenol / chloroform extract, ethanol / NaCl precipitate and resuspend in 25ul DDW
Transcription Reaction:
Reagent amt / reaction 5x transcription buffer 20ul 10 x DDT 10ul DDW 40ul rNTP 20ul RNasin 0.5ul T7RNA polymerase 1ul Add to 5ug DNA template. Mix reaction, adding enzyme (T7) last.
Incubate 37°C 90 minAdd equal volume phenol/chloroform (100ul). Vortex. Spin 13000 rpm 2 min
Add supernatant to new eppendorf and ethanol precipitate 2.5 vol ethanol and 0.3M NaCl final conc..
Resuspend 25 ul TE
Run 3ul RNA on Northern gel to check the template.
Northern
| - | 1.5g agarose in 130ml DDW. Boil to dissolve agarose and add 15ml 10 x MOPS. Cool and add 15ml formaldehyde. Add 3ul RNA and 7ul Sample buffer. Sample buffer (90ul total) = 50ul deionized formamide, 20ul formaldehyde, 10ul 10 x MOPS and 10ul 400 ug / ml Ethidium Bromide |
| - | Heat RNA 65oC 10 min |
| - | Run at 100v 2 hrs in 1 x MOPS. The sample should contain 0.2-1.0ug RNA. |
Translation
We use Promega rabbit reticulocyte lysate. S35 methionine is DuPont translation grade (5mCi/mmol)
Heat RNA 60°C 5 min, ice and prepare reaction cocktail. The lysate can be refrozen once. Only prepare a cocktail with the amount required.
Lysate 200ul
DDW 55 ul
RNasin 4 ul
AA-met 6 ul
35S met 25 ul
For example:
Add 50ul reaction mix to 5ul RNA
Incubate 30°C 60 min. Freeze - 20oC
Binding to purified GEX fusion proteins or antisera
Combine 15ul lysate with 5ul fused protein (2mg/ml) or 5ul antisera in 0.5ml PLC basic lysis buffer (see Appendix)
Incubate ice 1 hr
Swell 2mg Glutathione agarose beads (Sigma G-4510) or 2.5mg protein A sepharose (for antisera, P-3391, Sigma) per sample in PLC buffer / 10mM DTT and resuspend beads 0.1 ml PLC buffer per sample. Add 0.1ml of beads per sample and incubate on a wheel at 4°C for 1hr.
Add 100ul bead suspension / binding reaction and place of the wheel at 4oC for 1 hr
Pellet samples in ependorf for 30 sec and remove s/n to last 50ul with a blue tip. Add ice cold PLC -lysis buffer and vortex.
Repeat 3 x, then aspirate all buffer from beads with Gilson pipette
Add 40ul SDS sample buffer
Run on SDS PAGE gel.
Stain with Coomassie and destain. Photograph gel to check recovery of GST proteins or antibody heavy chain with beads.
Amplify (Amersham RPN 2106) 30 min, dry and autoradiograph
Testing protein-protein interactions with in vitro transcribed and translated proteins.
The two proteins should be translated such that one is labelled with 35S methionine and one is unlabelled. If binding is to be assayed by Immunoprecipitation, then the "bait" protein to which the precipitating antibody is directed should be unlabelled and the "target" protein labelled. Having the two proteins labelled is possible, but probably best avoided to keep the amount of label down, and in cases where "target" protein and "bait" proteins are the same size. A control with vector only as "bait" should be used to show non-specific binding of labelled proteins to the beads. If background is a problem, then blocking agents such as 5% skim milk powder or 0.8% BSA could be used. Preclearing the immunoprecipitation with protein A/G sepharose is also a possibility.
Prepare proteins separately by in vitro transcription and translation according to the standard Promega TNT reticulocyte protocol. Adjust total reaction volume so there is 12.5ul of each lysate per binding assay.
Add 12.5ul of each protein lysate to an eppendorf and mix by pipetting. Allow proteins to associate for 30 minutes at 4°C.
Add 200ul of cold immunoprecipitation buffer. Suitable buffers include basic PLC (with or without EGTA), RIPA buffer (with or without SDS) and PBS with 0.01% lubrol. Choice of buffer is dependant on the strength of interaction. Probably the gentlest conditions are PBS with lubrol. Be aware that some protein-protein interactions may be dependant on metal ions such as zinc, and these should be supplemented where appropriate.
Add antisera (5-10ul crude sera) and 2.5mg protein A/G sepharose. Incubate overnight at 4°C with gentle agitation.
Spin down beads and wash three times in 1ml of immunoprecipitation buffer. The first wash should be carried out in the hood and the radioactive waste discarded appropriately.
Resuspend beads in 30ul SDS sample buffer and run 20ul on a polyacrylamide gel. Dry gel down and autoradiograph.
