Name: measuring protein binding to f-actin by co-sedimentation
Uploaded: 2017-05-18T12:30:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

이 연구는 National Institutes of Health Grant HL127711에 의해 AVK에 의해 지원되었습니다.

<ol>
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저자는 경쟁적인 금전적 이해 관계가 없다고 선언합니다.

액틴 동시 침강 분석은 단백질이 F- 액틴에 결합하는지 신속하게 결정할 수있는 직접적인 기술입니다. 약간의 변경을 가하면이 기법을 사용하여 상호 작용의 친 화성을 측정 할 수도 있습니다. 위의 프로토콜에서 제기 된 점 외에도 분석을 디자인, 실시 및 해석 할 때 다음 사항을 고려해야합니다. 관심있는 단백질 신선하게 준비되거나 동결 된 단백질을 분석에 사용할 수 있습니다. 동결 단백질이 사용되는 경우 동결이 F- 액틴 바인딩에 영향을 미치지 않는지 확인하기 위해 결과를 신선한 (결코 동결되지 않은) 단백질과 비교하는 것이 좋습니다. G- 액틴 소스 많은 pelleting 실험은 G-actin을 상대적으로 풍부하기 때문에 근육에서 분리하여 사용합니다. 포유류에서 알파, 베타 및 감마와 같은 세 가지 주된 액틴 이소 타입이 있습니다 (90 % 이상의 서열 식별 기호ty). 그럼에도 불구하고 isotypes 12 , 13 에는 기능적 차이가 있습니다. 가능한 경우, 결합 분석에서 사용되는 G- 액틴 이소 타입 은 생체 내 아이소 타입과 일치해야합니다. 예를 들어, 골격근에서 발현 된 단백질을 테스트 할 경우 알파 - 굴지가 최선의 선택입니다. 섬유 아세포에서 발현되는 단백질을 검사 할 경우 베타 - 액틴 (beta-actin)을 권장합니다. Phalloidin 사용 Phalloidin은 F-actin과 결합하기 때문에 일부 F-actin 결합 단백질 ( 예 : phalloidin 블록 cofilin과 actin 필라멘트의 결합) 14의 결합을 방해하거나 차단할 수 있습니다. 따라서, phalloidin은 조심해서 사용해야하며 가능한 경우 phalloidin으로 처리하지 않은 샘플과 비교합니다. 높은 배경 단백질이 F- 액틴의 부재하에 침전되는 것은 드문 일이 아닙니다 ( 그림 1A , F- 액틴 펠렛 샘플 없음에스). 그러나 높은 수준의 백그라운드 침전은 진정한 액틴 동시 침강을 가려 낼 수 있으며 불가능하지는 않더라도 단백질이 F- 액틴에 결합하는지 또는 상호 작용의 친화력을 측정 하는지를 결정하는 것을 어렵게 만듭니다. 폴리 디카 놀을 반응 완충액 (단계 4.1)에 첨가하면 배경을 상당히 감소시킬 수 있고 쉬운 해결책이다. 그것이 배경을 감소시키지 않는다면, 반응 완충액, 소금 농도 및 / 또는 배양 온도를 조정하는 것이 도움이 될 수 있습니다. 제본 곡선 결합 곡선을 생성하기 위해서는, 일련의 반응에 대해 관심있는 단백질 또는 F- 악틴의 농도를 변화시키는 것이 필요하다. 실제로, 고정 농도에서 F- 액틴을 유지하고 관심있는 단백질의 농도를 변화시키는 것이 더 쉽고 바람직하다. pelleting 분석에서 고정 농도 ( 예 : 2 μM)에서 F-actin을 유지하면 고농도의 F-actin 농도에서 비특이적 트래핑이 제한되어 예방됩니다F-actin 농도가 낮은 (&lt;0.5 μM) 경우 해중합. Phalloidin을 사용하여 해중합을 막을 수는 있지만, 이것은 시스템에 잠재적 인 복잡 요인을 유발합니다 (3.3 단계 이상 참조). 고정 농도에서 F - 굴지 유지는 또한 샘플을 통해 F - 액틴 펠렛을 비교 (그리고 정상화)하고 실패한 실험 ( 즉, F - 액틴 펠렛은 매우 가변적이어서 농도에 따른 분석을 막음)을 식별 할 수 있습니다. 마지막으로, 고정 농도에서 F- 굴지를 유지하면 액틴 필라멘트에 대한 결합이 협동인지를 결정할 수 있습니다 ( 그림 1C ). 포화 바인딩 모든 결합 실험에서와 같이, F- 액틴에 대한 결합이 포화되어 있고 단백질 + F- 액틴 플러스의 농도가 중요합니다 ( 그림 1C ). 고원이 없으면 정확한 해리 평형 상수를 계산할 수 없습니다. 따라서,시험 할 희석 시리즈를주의 깊게 계획하고 항상 더 높은 농도의 단백질을 포함하는 것이 중요합니다 ( 예 : 예상 Kd보다 적어도 5 ~ 10 배 이상). 바인딩 분석 측정 된 해리 상수가 결정적이기 위해서는, 관심 단백질에 대한 F-actin상의 결합 부위의 농도가 친 화성보다 훨씬 낮아지는 F- 액틴 농도를 사용하여 분석을 수행해야합니다. 이 기준이 충족되었는지 여부를 확인하려면 Bmax에서 결합 사이트의 농도를 추정하십시오. 예를 들어 [F- 액틴]이 2 μM이고 Bmax = 0.5 인 경우 [결합 부위]는 약 1 μM입니다. K d 는 [결합 부위]보다 적어도 5 내지 10 배 더 커야한다. 측정 된 K d 가 [결합 부위]와 동일한 정도의 크기라면, 관찰 된 결합 곡선은 고친 화성 결합 si의 적정을 나타내는 것이 가능하다tes보다는 진정한 구속 isotherm. 이것이 관찰되면 정확한 친화도를 측정하기 위해 10 배 낮은 F-actin 농도를 사용하여 분석을 반복하십시오. 고친 화성 상호 작용의 경우, phalloidin 안정화 (단계 3.3)는 정확하게 친화도를 측정 할만큼 충분히 낮은 F- 액틴 농도를 달성하는 데 필요할 수 있습니다. 마지막으로, 분석을 수행하고 평가할 때 연구자가 알아야 할 공동 침강 분석의 근본적인 한계가 있습니다. 가장 중요한 것은 공 침강 분석이 진정한 평형 상수를 생성하지 않는다는 것입니다. 원심 분리 과정에서 결합 생성물 ( 즉, 단백질 + F- 액틴)이 반응물과 분리되면 생성물은 해리되어 새로운 평형을 생성 할 수 있습니다. 결과적으로, co-sedimentation assay는 낮은 친화도의 상호 작용을 오산하거나 잘못 검출 할 수 있습니다. 많은 액틴 결합 단백질은 F- 액틴에 대해 낮은 ( 즉, 마이크로 몰러) 친화력을 가지므로, 부정적인 결과 ( <e즉, 검출 할 수없는 결합)이 반드시 단백질이 F- 액틴에 결합하지 않는다는 것을 의미하는 것은 아니다. 대안으로, TIRF 현미경 기반의 단일 필라멘트 결합 분석법은 해리 상수를 결정할 때보다 민감하고 정확합니다 (이 기술에 대한 리뷰, 참고 자료 15,16 참조). 이러한 제한에도 불구하고, pelleting 분석은 대부분의 연구자의 수단에 속하며 단백질이 F-actin에 결합하는지 여부와 상호 작용의 친 화성을 측정 할 수있는 효과적인 도구입니다.

액틴은 운동성, 수축, 접착 및 형태학을 포함한 여러 세포 과정에서 중요한 역할을하는 필수적인 세포 골격 단백질입니다 1 . 액틴은 단구 구형 액틴 (G- 액틴)과 중합 된 사상 액틴 (F- 액틴)의 두 가지 형태로 존재합니다. 세포 내에서 F- 액틴 조직은 액틴 필라멘트 2 , 3 , 4 의 핵 형성, 성장, 가교 결합 및 분해를 조절하는 많은 단백질 모음에 의해 제어됩니다. 그러나 액틴 네트워크 구성을 조절하기 위해 여러 가지 액틴 결합 단백질이 함께 작용하는 방식은 아직 많이 알려져 있지 않습니다. 단백질 - 단백질 상호 작용의 측정은 단백질이 생화학 적 수준에서 세포 행동에 미치는 영향을 이해하는 중요한 접근법입니다. 많은 다른 assays는 정제 된 단백질 간의 상호 작용을 감지하는 데 사용할 수 있습니다.가용성 단백질에 대한 일반적인 접근법에는 풀다운, 형광 편광, 등온 적정 열량계 및 표면 플라스 몬 공명이 포함됩니다. 중요한 것은, 이들 모든 분석법은 단백질이 용해 될 것을 요구하기 때문에, F- 굴지와 같은 고분자 필라멘트 단백질을 사용하기에 적응하기가 어렵습니다. 여기서 우리는 단백질 또는 단백질 도메인이 F- 액틴에 결합하는지 여부를 결정하고 상호 작용의 친화도를 측정하기위한 기술인 액틴 동시 침강 (actin co-sedimentation) 또는 펠렛 (pelleting) 분석을 설명합니다. 액틴 펠릿 법은 초 원심 분리기를 제외하고 특수 장비가 필요없는 비교적 간단한 기술입니다. 모든 시약은 기본 생화학 지식을 전제로하여 만들거나 구입할 수 있습니다. F - 굴지에 바인딩되면, 분석은 겉보기 친화력 ( 즉, 해리 평형 상수) 5 측정하는 데 사용할 수 있습니다. 또한 일단 친화력이 확립되면, 펠릿 화 분석법는 관심 단백질 ( 예 : 번역 후 변형, 돌연변이 또는 리간드 결합)이 F- 액틴 6 과 어떻게 상호 작용 하는지를 측정하는 유용한 도구입니다. 이 기법은 분석을 시도하기 전에 연구자가 알아야 할 한계가 있습니다 (토론 참조).

<table><tbody><tr><td>Sorvall MTX 150 Micro-Ultracentrifuge</td><td>ThermoFisher Scientific</td><td>46960</td><td></td></tr><tr><td>S100-AT3 rotor</td><td>ThermoFisher Scientific</td><td>45585</td><td></td></tr><tr><td>Ultracentrifuge tubes - 0.2 mL</td><td>ThermoFisher Scientific</td><td>45233</td><td></td></tr><tr><td>Actin, rabbit skeletal muscle</td><td>Cytoskeleton</td><td>AKL99</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A8531</td><td></td></tr><tr><td>Polidicanol (Thesit)&amp;nbsp;</td><td>Sigma</td><td>88315</td><td></td></tr><tr><td>Phalloidin</td><td>ThermoFisher Scientific</td><td>P3457</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>ThermoFisher Scientific</td><td>R0862</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Sigma</td><td>A2383</td><td></td></tr><tr><td>Imidazole</td><td>Fisher Scientific</td><td>O3196</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>M33</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217</td><td></td></tr><tr><td>Ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma</td><td>3779</td><td></td></tr><tr><td>Odyssey CLx Imaging System</td><td>LI-COR</td><td></td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250 Dye</td><td>ThermoFisher Scientific</td><td>20278</td><td></td></tr><tr><td>Colloidal Blue Staining Kit</td><td>ThermoFisher Scientific</td><td>LC6025</td><td></td></tr></tbody></table>

measuring protein binding to f-actin by co-sedimentation

세포 내에서 필라멘트 액틴 (F- 굴지) 조직은 액틴 핵 생성, 성장, 가교 결합 및 / 또는 분해를 조절하는 다수의 액틴 결합 단백질에 의해 조절된다. 이 프로토콜은 단백질 또는 단백질 도메인이 F- 액틴에 결합하는지 여부를 결정하고 상호 작용의 친 화성 ( 즉, 해리 평형 상수)을 측정하는 기술 - 액틴 동시 침강, 또는 펠렛 화 - 분석을 설명합니다. 이 기술에서 관심 단백질은 먼저 용액 내 F- 굴지와 함께 배양됩니다. 이어서, 차동 원심 분리를 사용하여 액틴 필라멘트를 침전시키고, 펠릿 화 된 물질을 SDS-PAGE로 분석한다. 해당 단백질이 F- 액틴에 결합하면 액틴 필라멘트와 함께 침강합니다. 결합 반응의 생성물 ( 즉, F- 액틴 및 관심 단백질)은 상호 작용의 친화력을 결정하기 위해 정량화 될 수있다. 액틴 펠릿 화 분석법은관심 단백질이 F- 액틴에 결합하고 리간드 결합과 같은 단백질의 변화가 F- 액틴과 어떻게 상호 작용하는지 평가할 수 있습니다.

우리는 co-sedimentation assay에서 F-actin에 결합하는 α-catenin homodimer를 검사했다. 과거의 실험은 F - 굴지에 대한 αE - catenin 동종 이합체의 친화도가 약 ​​1 μM이고 Bmax가 1 11 근처임을 보여 주었기 때문에, 우리는 낮은 농도의 F- 액틴 (0.2 μM보다는 2 μM; 토론). 0.2 μM이 임계 농도보다 낮기 때문에 phalloidin을 첨가하여 토끼 골격근 G-actin (단계 3.3)에서 중합 된 F-actin을 안정화시켰다. 증가하는 농도의 α-catenin homodimer (0.125-12.0 μM)를 0.2 μM F-actin의 존재 또는 부재 하에서 배양 하였다. 샘플을 원심 분리하고 결과물 인 펠렛을 분석 하였다 ( 그림 1A ). 예상대로 α-catenin 동종 이합체가 배경 위에 F- 액틴과 함께 침강 ( 그림 1A , F- 액틴 펠렛 샘플을 F-AC와 비교 함)주석 펠릿 샘플). BSA는 음성 대조군으로 수행되었다 ( 그림 1B ). 바운드 단백질을 정량하고 자유 단백질에 플롯하여 상호 작용의 친 화성을 계산했습니다 ( 그림 1C ). 플롯 된 데이터는 Hill 방정식에 가장 잘 맞습니다. 계산 된 Kd는 2.9 μM, Bmax는 0.2, Hill 계수 (h)는 4.8이었다. 따라서, αE - catenin 동종 이합체는 이전의 연구 (~ 1.0 μm의 대 2.9 μm의)와 일치, 낮은 micromolar 친화력과 협조 F - 굴지에 바인딩 11 . <img alt="그림 1" src="/files/ftp_upload/55613/55613fig1.jpg" /> 그림 1 : 고속 F- 액틴 동시 침전 분석. ( A ) 증가하는 농도 (0.125-12.0 μM)의 αE-catenin homodimer를 phalloidi로 안정화 된 0.2 μM F-actin (왼쪽 패널) 또는없는 (오른쪽 패널) 배양 하였다엔. 그들은 RT에서 30 분 동안 배양하고 원심 분리시켰다. 총 (출발 물질의 7.5 %) 및 펠릿 화 된 물질 (펠릿 화 된 물질의 50 %)을 SDS-PAGE로 분리하고 쿠마시 염료로 염색 하였다. ( B ) 4 μM BSA를 음성 대조군으로 사용 하였다. F- 악틴이있는 (+) 또는없는 (-) 총 및 펠릿 샘플을 SDS-PAGE로 분리하고 쿠마시 염료로 염색 하였다. ( C ) 자유 αE - catenin (μM)에 대해 A로부터 결합 된 αE-catenin (μM / μM actin)을 나타내었고, Hill 방정식 (적색선)에 적합 하였다. K d , B max 및 Hill 계수 (h)가 나열됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/55613/55613fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/55613/55613fig2.jpg" /> 그림 2 : 액틴 펠릿 화 정량화 </strong&gt; - 순서도. 이 도식은 총계 및 펠렛 샘플 ( A , CE ) 및 정량화에 사용되는 표준 곡선 ( B )의 예와 함께 섹션 5의 주요 단계를 간략하게 설명합니다. 5.13 단계 : 1 ) 총 시료 (A)에서 관심 단백질의 양을 측정합니다. 2 ) 단백질 강도 (B) 대 밴드 강도를 플로팅하여 표준 곡선을 생성합니다. 3 ) F - 굴지 ( C )와 함께 침전 된 관심 단백질의 양을 측정합니다. 4 ) F - 굴지 ( D )의 부재에서 pelleted 관심 단백질의 양을 측정합니다. 5 ) F를 액틴에 결합하는 단백질의 양을 결정하기 위해 C에서 D를 뺍니다. 6 ) 각 펠렛 (E)에서 F- 액틴의 양을 측정하고, 샘플 당 F- 액틴의 평균 양을 계산하고, 평균으로 각 샘플을 나눕니다 (아래 숫자는 비율을 나타냄). 7 )각 샘플에 대해 펠렛의 차이를 조정하기 위해 결합 된 단백질의 양 (단계 5에서 계산)을 F- 액틴 펠릿 비율 (단계 6에서 계산)로 나눕니다. 8 ) 표준 곡선 ( B )를 사용하여 각 샘플에서 표준화 된 결합 단백질의 양 (질량)을 계산합니다 (단계 7). 9 ) 결합 곡선을 만들기 위해 유리 단백질과 결합 단백질의 농도를 결정하십시오. <a href="http://ecsource.jove.com/files/ftp_upload/55613/55613fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

Measuring Protein Binding to F-actin by Co-sedimentation

이 프로토콜은 필라멘트 액틴 (F - 굴지)과 함께 공동 침강하는 단백질의 능력을 테스트하는 방법을 설명하고, 바인딩이 관찰되면 상호 작용의 친화력을 측정하는 방법을 설명합니다.

공동 침강에 의한 F-actin에 대한 단백질 결합 측정

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

measuring-protein-binding-to-f-actin-by-co-sedimentation

Research

JoVE Journal

Biochemistry

16.4K 조회수.  University of Pittsburgh School of Medicine.  이 프로토콜은 필라멘트 액틴 (F - 굴지)과 함께 공동 침강하는 단백질의 능력을 테스트하는 방법을 설명하고, 바인딩이 관찰되면 상호 작용의 친화력을 측정하는 방법을 설명합니다. 

비디오: Measuring Protein Binding to F-actin by Co-sedimentation

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

단백질 - 단백질 상호 작용을 연구하는 도구로 형광 이방성

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

연구

생화학

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

작은 올리고머 형성 막 단백질의 침전 평형 : 펜타 머 안정성에 히스티딘 양성자의 효과

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

화학

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

생물학

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

셀 연락처에서 단백질 단백질 상호 작용의 형광 변동 분광학 분석 결과

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

단백질 기능에 2DE - 젤 지점에서 만성 림프 구성 백혈병에 HS1에서 배운 레슨

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

의학

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

형광 크기 배제 크로마토그래피를 통한 멤브레인 단백질 품질의 신속한 평가

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

글루칸 포스파타제의 기질 결합을 측정하기 위한 Concanavalin A 기반 침강 분석

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

상업적으로 이용 가능한 단백질을 사용한 액틴 기반 운동성의 재구성

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

생체공학

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ 중합 분석 실험 : 간단한 프로토콜 및 고려 사항

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

단백질 상호 작용의 Live Cell Single-Molecule Imaging-Based 정량화

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

저자 스포트라이트: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

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Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...