生物化学技术

The multiple copies of the HA epitope present in the HAT tag can be detected by the mouse monoclonal antibodies 12CA5 (Boehringer) and 16B12 (MMS101R; BAbCO, Richmond, California) (see Table 1). On Western blots against yeast protein, these antibodies often recognize a single cross-reacting ...

This protocol describes our method for preparing cells for immunofluorescence, in which all incubations and washes are performed in microtiter dishes. Two dishes can be processed simultaneously. The last step is to transfer each sample to a well on a polylysine coated 8-well slide for visual ...

In order to decrease the amount of nonspecific staining, it is often necessary to preabsorb primary and secondary antibodies to yeast cells lacking the antigen prior to use. A 1:1 mixture of fixed yeast whole cells and spheroplasts are used for this purpose.Grow cells in 200 mls of YPD at 30oC to an OD 600 of 1. Add ...

Fix cells in 2% formaldehyde in PBS/pH 7.4 for 15 min. at 20oC. 2% formaldehyde is made up fresh prior to use by dissolving the appropriate amount of EM grade paraformaldehyde (Prill form, from Electron Microscopy Sciences) in PBS and heating on a hot plate in the hood (setting of 5-6) until the aldehyde goes into s ...

Wash XCell box, blotting module and trays with soap and water before proceeding.Reagents:Transfer buffer25 mM Tris HCl, 190 mM Glycine10% MethanolBlocking buffer5% non-fat dry milk in TBS-TWEEN 20or3% BSA in TBS.TBS-TWEEN 2025 mM Tris HCl 137 mM NaCl0.05% TWEEN-20Vectastain ABC kitSpeci ...

Whole mount in situ hybridization with digoxygenin probes1. Probe labelling (DNA) DNA template (100 ng - 1 µg ) in dH20 5 µl pd(N)6 (10 mg/ml) 10 µl Mix template and hexamer, boil 5 min, chill on ice 3 min then add : 10 X Hexanucleotide buffer (or Vial 5 of Genius kit) 2 µl dig-dNTP mix ( Vial 6 of Genius kit ) 2 µl Klenow 1 µl Incubate at 37 C o/n Add 1 ...

Procedure:(This protocol uses anti-XKCM1 for immunodepletion.) Put 25 ul of Bio-Rad Affi Prep bead slurry into two 0.5 ml tubes labeled IgG and XKCM1. Wash beads 3X with 0.5 ml TBST each wash. Add Rb IgG (4 ug) or anti-XKCM1 Gly (4 ug) and bring volume to 100 ul total. Bind antibody to beads at 4 deg.C for 1 hr 15' on rotator. Make su ...

Problems:The main problem with immunodepletion of crude CSF extracts is that they activate during or soon after immunodepletion. Empirically, the following modifications have allowed us to succesfully immunodeplete and characterize three different proteins: a) During extr ...

Antibody Staining of Larval Imaginal Discs1. Dissect discs in PBS.2. Fix in 3% formalin in PBS for 15-30 minutes at room temperature.3. Wash for 30 minutes in 3 changes of PBT.4. Incubate with primary antibody in PBTN for 1 hr. to overnight. 5. Wash tissue for 30 min. to overnight in PBS.6. Incubate for 1 hr. to overnight ...

Antibody Staining of Ovaries1. Dissect ovaries in EBR2. Transfer ovaries into an Eppendorf tube and add 100 µl devitellinizing buffer and 600-µl heptane3. Agitate gently for 10 minutes4. Remove solution and rinse ovaries with PBS 3 times, then wash 3 times for 10 minutes each5. It may help antibody p ...

Laboratoire de Biologie Cellulaire Station de Genetique et Amelioration des Plantes Institut National de la Recherche Agronomique 78026 Versailles Cedex FRANCEThis protocol is based on excerpts of the following papers :Bouchez D, Camilleri C, Caboche M (1993). A binary vector based on ...

Antibody Staining of Imaginal DiscsFrom DIS- Thom Kaufman's lab1. Dissect tissues in Tri-PBS, gather in a 1.5 ml tube of Tri-PBS on ice(at least 10 animals can be processed in one tube). Tear larvae in half and invert the anterior portion; remove all the gut tissue from the mutant larvae by cutting at the esopha ...

ARABIDOPSIS TRANSFORMATIONSeed Sterilization. Sterilize WS (Wassilewskija) seed for 10 min in a solution of 50% Chlorox with 0.1% SDS in ependorf tubes. Rinse 3 to 5 times with dH20. Dry thoroughly on sterile filter paper. Root cultures. Place 35 seed into each of 35 250 ml Belco flasks with 50 ml B5 medium. P ...

Arabidopsis Seed CollectionLuca Comai has devised a simple seed collector suitable for high density use (many plants in a small space). It is effective, very cheap, and easy to construct. The instructions for constructing it are given below. They may sound complicated but the collector is really ...

Cuticle preparations-Collect embryos on apple juice-agar plates for suitable time period from a well-stocked cylinder or cage. Allow to age for 24-36 hr at 25o.-rinse unhatched larvae into a nytex screen. Dechorionate in 3% bleach until embryos float to the surface (1-5 min). Don't worry about over ...

I. MEDIUMA. S2, S2C, Sundefined, S2R+, S3, l(2)mbn, Kc(167), & DL2 cells: Schneider's/ 10% FBS/ PS450 ml Schneider's medium (Gibco #11720-034)(pour off 50 ml into Falcon to save as serum-free~undefined*50_ml_Fetal_Bovine_Serum_~AJRH_~512103~F78P~B_~F_Heat_Inactivated_~Aaliquoted_in_~F20~B5_ml_1~I100_Penicillin~FStreptomycin_~AGibco__...

In Situ HYBRIDIZATION TO TISSUE SECTIONS(Kornberg et al., Cell 40:45, 1985))PARAFFIN SECTIONSPreparation of embryos (¾10 hr) for paraffin sections Wash embryos off collection plates onto a nylon screen with 0.4% NaCl/0.3% Triton X-100. Rinse well to remove all food and yeast. Blot well to remove e ...

IMMUNOHISTOCHEMISTRY ON WHOLE MOUNT EMBRYOS(Paul MacDonald)Dechorionate directly on apple juice/agar plates with 50-60% chlorox; embryos will float to the surface after 2-3'. Collect embryos on a Millipore device with a nitex filter; rinse well with 0.1% Triton X-100 from a squirt bottle. T ...

IMMUNOHISTOCHEMISTRY ON SCHNEIDER LINE 2 CELLS 1. Plate 400ml of a 105 cells/ml suspension into 8-chamber slides (plastic). Allow the cells to settle and attach for a couple hours. 2. Gently aspirate off the media and rinse cells 2-3X in PBS. 3. Rinse 1X in 100mM NaHPO4, pH 7.2-7.4 (diluted from 1M stock). 4. Fix 15-3 ...

DROSOPHILA RNA PREP(Goodman Lab) Stock Solutions 3M NaOAc, pH 5.2 with HAc (MW=82) 6M Guanidine Hydrochloride in 0.1M NaOAc, pH 5.2 (MWGuHCl=95.54) 5.7M Cesium Chloride in 0.1M NaOAc, pH 5.2 (MWCsCl=168.37) Procedure 1. Brush embryos onto teflon pan with ddH2O and filter adults on nitex. 2. Rinse well with d ...