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Detection of Western blots using fluorescent labeled secondary antibodies can allow for more accurate quantification and two-color multiplexing using primary antibodies from different species. Phosphorylation and total protein levels may be determined on the same membrane, and regulation of different targets at different molecular weights can be examined simultaneously. There can also be significant long-term advantages in terms of digital data storage as well as the reduction of costs for chemiluminescent substrates, x-ray film, and darkroom facilities.

Western blot analysis of COS cells extracts.

Western blot analysis of extracts from COS cells, untreated or treated with either U0126 (#9903) (10 µM for 1h) or TPA (#4174) (200 nM for 10m), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb (#4370) and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb (#9107).

Western blot analysis of Raw264.7 cells extracts.

Western blot analysis of extracts from Raw264.7 cells, untreated or LPS-treated (1 µg/ml for 6 h), using iNOS Antibody (#2977).

Many CST antibodies are being tested by Western blot using near-infrared dye-conjugated secondary antibodies and a slightly modified immunoblotting protocol. We have found that omitting Tween-20 during the blocking step is the only significant change necessary. Full details can be found in our Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In BSA) and our Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In Milk). We have also found that prestained standards such as our Prestained Protein Marker, Broad Range (Premixed Format) (#7720) are autofluorescent at near-infrared wavelengths.

Two-color Western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment. Some antibody pairs may not work together due to overlapping epitopes, interference caused by primary and/or secondary antibodies, or incompatibilities in primary antibody incubation buffers.



  1. コード番号:   (例) 4060
  2. バイアル記載のLot No.:   (例) 1
  3. バイアル記載のReference Date:   (例) 07/2008
  4. 納入日:   (例) 10/2008
  5. 保存温度:  4℃  -20℃  -80℃  その他
  6. 小分け保存しましたか?: Yes  No


  1. 細胞: Cell Line  Primary Cells  Whole Tissue  その他
  2. : Human  Mouse  Rat  その他
  3. 処理薬物:   (例) EGF
  4. 処理薬物の濃度:   (例) 5 mM
  5. 薬物の処理時間:   (例) 1, 3, 5 min
  6. ポジディブコントロール:   (例) Jurkat (Calyculin A処理)
  7. 可溶化バッファー:
     SDS Sample Buffer  CST Cell Lysis Buffer  CST Chaps Buffer
     RIPA Buffer  その他
  8. 可溶化バッファーに添加したフォスファターゼ阻害剤 (あれば):   (例) NaF
  9. サンプルは超音波処理しましたか?Yes No
  10. アプライしたタンパク量 (ug/lane) : 20μg  その他
  11. ライセートを調製してからの期間:   (例) 2 weeks
  12. ライセートの保存温度: 4℃  -20℃  -80℃  その他


  1. アクリルアミドゲルの濃度 (%) :   (例) 10%
  2. 転写膜: Nitrocellulose  PVDF  Nylon  その他
  3. 転写膜のポアサイズ:   (例) 0.45 μm


  1. ブロッキングバッファーの組成:   (例) 5% Nonfat dry milk/ TBST
  2. ブロッキングの温度と時間:   (例) 室温/ 1 hr


  1. 希釈率:   (例) 1:1000
  2. 希釈バッファーの組成:   (例) 5% Nonfat dry milk/ TBST
  3. 反応温度と反応時間:   (例) 4℃/ 一晩
  4. 洗浄バッファーの組成と一次抗体反応後の洗浄時間:   (例) TBST/ 5 min×3


  1. 由来:   (例) goat-anti rabbit IgG/ CST
  2. 希釈率:   (例) 1:2000
  3. 希釈バッファーの組成:   (例) 5% Nonfat dry milk/ TBST
  4. 反応温度と反応時間:   (例) 室温/ 1 hr
  5. 洗浄バッファーの組成と二次抗体反応後の洗浄時間:   (例) TBST/ 5 min×3


  1. 検出システム: HRP  AP  その他
  2. 検出システムの製造元メーカー名: 
  3. 画像取得の設定について: 
  4. 問題点: 詳細をご記入ください。実験データの添付も可能です。

添付ファイル  (5MBまで)




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