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問題	推奨される解決策
ネガティブコントロール（IgGまたはモックによるIP）サンプルのバックグラウンドが高い	過剰な抗体によって標的以外に結合が生じている：抗体の濃度を最適化します。ビーズへの非特異的結合：ビーズの事前洗浄を実施するか、ブロッキング剤をビーズに添加します。クロマチンの断片化が不完全である：断片化工程を最適化し、クロマチンの長さを200～1,000 bpにします。細胞や組織の種類ごとに、断片化を最適化します。シリコン処理したチューブ、または低吸着チューブを用います。試薬が汚染されている：すべての試薬が新しく調製されており、夾雑物を含んでいないことを確認します。洗浄回数を増やします。サンプルに核酸が混入していないかどうかを判定するために、「DNAなし」でPCR反応を実施します。
「DNAなし」のPCR反応でシグナルが認められる	PCR専用のピペットを用い、PCRのセットアップ前にピペットをUV照射します。 ChIP、DNA精製、PCR反応のセットアップを、それぞれ別の3室／区域で、専用のピペットを用いて実施するか、反応をフード内でセットアップします。ボトルに入った水など、事前に包装された水の使用は避けます。UV光源を備えたシステムから供給された新鮮なMilli-Q®水を用います。エアロゾル耐性ピペットチップを用います。 PCR増幅産物の入ったチューブは、ChIPまたはqPCR実験の実施予定場所付近で開封しないようにします。
インプットDNAで増幅しない	DNAサンプルは、抗体を加える直前に採取します。これを出発物質とみなします。インプットDNAで増幅が見られない場合は、架橋の反転が上手くいっていないか、キット以外の理由でPCRが作動していません。
DNAの回収率が低い	ChIP抗体が無効、または親和性が低い：ChIPで検証済みの抗体を用いていることを確認しましょう。ChIP抗体を用いている場合は、抗体のインキュベーション時間を延長します。 ChIP抗体が不十分である：通常、ChIP抗体は1～10 µgで十分です。しかし、抗体の必要量は、標的タンパク質の相対的な発現量や、標的に対する抗体の親和性によって異なる場合があります。初期サンプルが不十分である：架橋前に、細胞数判定用のプレートを別途作製します。1反応あたりの細胞数を再評価します。特に発現量が少ない標的を検出したい場合には、細胞数を増やします。細胞溶解が不完全で、断片化が無効：パラメータを変え、クロマチン回収率に対する影響を評価することで、細胞溶解と断片化のステップを最適化します。細胞溶解を改善するため、ダウンス型ホモジナイザーやガラスビーズなどの物理的な力を使います。断片化のステップを最適化し、泡立たないようにします。過剰な架橋：ホルムアルデヒド中で長時間インキュベーションすると、ChIP抗体による認識に必要なエピトープが遮蔽されてしまう恐れがあります。モノクローナル抗体を用いている場合、これは特に問題となります。過剰な架橋によって、超音波処理に耐えられる複合体が形成されてしまう場合もあります。架橋のステップを最適化します：ホルムアルデヒドの最終濃度を1%とし、実験を進める前に、最も有効な架橋時間を決める必要があります。架橋が不十分：架橋が不十分であると、プロトコルのその後のステップ中に標的タンパク質がDNAから解離してしまう場合があります。ヒストンやヒストン修飾の研究でない限り、標的タンパク質とDNAの結合を安定化するために、X-ChIPプロトコルを用いる必要があります。架橋時間を延長します。親和性が低い、またはビーズの品質が低い：プロテインG磁気ビーズは、マウスモノクローナル抗体を含め、さまざまな抗体に結合します。最も汎用性の高い抗体として、プロテインA/Gブレンド（カタログ番号：16-663）をお勧めします。 PCRプライマーのプライミング効率が低い：プライマーの効率を試験します。適切なコントロールを使い、必要に応じてプライマーを設計し直します。
大きなDNAだけが単離（プルダウン）される	固定や超音波処理の際に、過剰な固定や加熱を行うと断片化が阻害されることがあります。超音波処理時間が長くなるとエピトープが損傷する可能性があります。固定時間を短縮すると、超音波処理時間を短縮でき、より小さな断片が得られます。また、断片化が均一にできていることを確認するため、IP前に単離したDNAをゲル泳動し、約200～300 bpに断片化したDNAの濃いバンドが現れ、ゲル上に他のバンドがないか確認することを推奨します。固定反応はグリシンでは停止しません。
抗体を添加しないコントロールのほうがサンプルよりも多くのDNAが沈殿する	ネガティブコントロールのバンドを除去するには、以下を実施します：希釈した細胞ペレット懸濁液（2 mL）にSalmon Sperm DNA/Protein A Agarose-50% Slurry（製品番号：16-157）を80 µL加え、4℃で30分間振とうしながら事前精製します。細胞ライセートの事前精製の時間を長くしたり、回数を増やすことも可能です。インプットDNAを滴定し、NFAのバンド消失時間を見極めます。別の溶解手順を用います：細胞ペレットをプロテアーゼ阻害剤を含有する5 mM Pipes（pH8.0、85 mM KCl、0.5% NP40）200 µLに再懸濁します。氷上で10分間静置します。遠心分離によりペレットを回収します（5000 rpmで5分間）。プロテアーゼ阻害剤含有1% SDS、10 mM EDTA、50 mM Tris-HCl混合液（pH8.1）200 µLにペレットを再懸濁します。氷上で10分間インキュベートします。使用前に1～5% BSA–Chip希釈バッファーでSalmon Sperm DNA Agaroseをブロックします（室温で30分間攪拌します）。インキュベーション後、アガロースを遠心分離し、1% BSA/ChIPアッセイバッファーの上清を除去します。ChIPアッセイバッファーで1回洗浄し、実験を続けます。

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