Reference protocols

Shared reference workflows for WB, IHC, IF, IP, Co-IP, ELISA, and cell culture—same source as product-page Protocols; optimize per datasheet and pilot runs.

Antibody assays

Generic immuno-assay reference workflows (for antibody products); optimize per datasheet and pilot runs.

Western blot (WB)

  1. Prepare lysates with protease/phosphatase inhibitors; clarify by centrifugation; quantify protein (e.g., BCA).
  2. Resolve proteins by SDS-PAGE; load 20–40 µg per lane depending on target abundance.
  3. Transfer to PVDF; adjust transfer time for high molecular-weight targets (~250–300 kDa).
  4. Block with 5% BSA or non-fat milk for 1 h at RT (or overnight at 4°C).
  5. Incubate with this product at the recommended dilution overnight at 4°C (or 2 h at RT); wash with TBST.
  6. Incubate HRP-conjugated secondary antibody; wash; develop with ECL and capture images.

Immunohistochemistry (IHC)

  1. Deparaffinize and rehydrate sections; perform heat-induced epitope retrieval if required.
  2. Block endogenous peroxidase; block non-specific binding with serum/BSA.
  3. Incubate with this product overnight at 4°C; detect with appropriate HRP polymer or biotin–streptavidin system.
  4. Counterstain, dehydrate, mount; image under a light microscope.

Immunofluorescence (IF)

  1. Fix cells/slides; permeabilize if needed for intracellular targets.
  2. Block, incubate with this product, then fluorescently labeled secondary antibody (protect from light).
  3. Counterstain nuclei, mount with antifade; image by confocal/widefield microscopy.

Immunoprecipitation (IP)

  1. Preclear lysate (optional); bind antibody to beads; incubate with lysate at 4°C with rotation.
  2. Wash beads thoroughly; elute with SDS sample buffer and analyze by WB.

Co-immunoprecipitation (Co-IP)

  1. Lyse under mild non-denaturing conditions (e.g. 0.5–1% NP-40/Triton X-100 with protease/phosphatase inhibitors); clarify and keep an Input aliquot (~5–10%).
  2. Preclear with Protein A/G beads (recommended) for 30–60 min at 4°C; collect the supernatant.
  3. Pull down the bait with this product (bait/target antibody) for 2–4 h or overnight at 4°C, then add Protein A/G beads for 1–2 h (or pre-bind antibody to beads).
  4. Wash beads gently 4–6 times with cold lysis/wash buffer; remove supernatant carefully.
  5. Elute in 2× SDS sample buffer (boil 5–10 min); analyze by SDS-PAGE/WB for the bait and the candidate interactor (detect the partner with a separate antibody).
  6. Controls: isotype IgG Co-IP and Input; optionally a known interaction pair (positive) or bait knockdown/knockout (negative).

ELISA

  1. Coat plate, block, add standards and samples, wash.
  2. Incubate with this product or paired detection antibody; wash; add enzyme conjugate and substrate; read absorbance.

Cell culture

Reference cell-culture workflows (maintenance, thawing, passaging, and freezing); optimize using the complete/freezing media on this page, cell type, and pilot data.

Cell culture (routine maintenance)

  1. Prepare incubator, biosafety cabinet, and sterile plastics; pre-warm complete medium and buffers to 37°C.
  2. Use complete medium prepared per datasheet for this product; note lot numbers. Atmosphere: 37°C, 5% CO₂.
  3. Seeding reference: adjust to cell line and vessel; mix gently; minimize incubator door openings.
  4. Monitor morphology and confluence; renew medium about every 2–4 days as needed.
  5. Isolate contaminated cultures and follow lab SOP; record passage and medium-change dates.

Cell thawing & recovery

  1. Thaw as soon as practical after receipt; for interim storage use LN₂ vapor phase—avoid long-term storage at −70/−80°C only.
  2. Thaw the vial by gentle agitation in a 37°C water bath for ~1–2 min until a small ice chip remains; keep the O-ring/cap out of the water.
  3. Wipe with 70% ethanol, open in a hood, and transfer into ~9 mL pre-warmed complete medium prepared per datasheet for this product; add medium slowly while mixing to reduce DMSO/osmotic shock.
  4. Centrifuge at ~125 × g for 5–7 min (or equivalent low-speed condition); aspirate supernatant and gently resuspend in fresh complete medium.
  5. To avoid excessive alkalinity on recovery, pre-equilibrate flasks with complete medium in the incubator ~15 min (target pH ~7.0–7.6) before seeding.
  6. Count viability and seed into a T25 or T75; next day inspect attachment/debris and change medium if needed to remove residual cryoprotectant.

Cell passaging

  1. Inspect cultures; passage at sub-confluence (do not wait for a full monolayer). Split reference: typically 1:3–1:6 (adjust to confluence). Seeding reference: adjust to cell line and vessel.
  2. Aspirate spent medium. Briefly rinse with Ca²⁺/Mg²⁺-free PBS or a small volume of trypsin-EDTA to remove residual serum (trypsin inhibitors).
  3. Add enough 0.05–0.25% trypsin or trypsin-EDTA to cover the monolayer; observe under an inverted microscope until cells round and disperse (often 5–15 min). Do not bang the flask while waiting; place at 37°C if detachment is slow.
  4. Add 2–3 volumes of serum-containing complete medium prepared per datasheet for this product to neutralize; pipette gently, count, and record passage.
  5. Seed new vessels at the target split; incubate at 37°C, 5% CO₂; log passage ratio and date.

Cell cryopreservation

  1. Harvest healthy sub-confluent adherent cells; detach/neutralize/count and plan cryovial numbers.
  2. Prepare freezing medium: 90% complete medium + 10% DMSO (optimize per SOP); keep chilled. (ATCC product sheets for related RIN lines often use complete medium + 5% DMSO; labs may optimize between 5–10% DMSO.)
  3. Pellet cells, resuspend in freezing medium to the recommended density (often ~1×10⁶–5×10⁶ cells/mL; optimize per line).
  4. Aliquot into labeled cryovials (SKU, designation, passage, date); cool via controlled-rate freezer or −80°C isopropanol box, then transfer promptly to LN₂ vapor phase.

Small-molecule inhibitors

Reference workflows for small-molecule inhibitors (dissolution, cell treatment, storage); optimize concentration and timing per cell model and pilot data.

Dissolution & stock prep

  1. Choose solvent per solubility (often DMSO); prepare concentrated stocks of this product and aliquot to limit freeze–thaw.
  2. Store stocks at ≤ −20°C; equilibrate before use and briefly centrifuge.
  3. Dilute into medium to the working concentration; keep vehicle ≤0.1% when using DMSO and include vehicle controls.

Cell treatment (reference)

  1. Seed cells to an appropriate density; treat after attachment (or per suspension assay window).
  2. Add this product working solution, mix gently; optimize exposure (1–72 h) for the chosen readout.
  3. Harvest for proliferation, apoptosis, pathway, or functional assays; record dose–response.

Storage & handling

  1. Protect solids and stocks from light and moisture.
  2. Follow chemical PPE and waste disposal SOPs.
  3. For research use only (RUO); not for clinical/diagnostic use.

Looking for a deep-dive note? See phospho-antibody purification, or return to technical support.