Immunoprecipitation: Difference between revisions
Created page with "This is an original excerpt from MHE's digital labbook from 25 April 2025. {{Protokoll-Box}} = Immunoprecipitation = - THP-1 Cells (WT, PPM1F KO (clone D7), PPM1F KO + PPM1F WT (clone C9), PPM1F KO+ PPM1F D360A (clone A5)) were count and 3x10<sup>6</sup> cells were lysed using 1000 µl RIPA lysis buffer (10 µg/ml Aprotinin,10 µM Benzamidin, 5 µg/ml Leupepin, 1 µM PMSF, 10 nM pNPP, 2 µM cytochalasinD, cpd26, 1x Phosstop) - Cells were vortexted, s..." |
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This is an original excerpt from MHE's digital labbook from 25 April 2025. | This is an original excerpt from MHE's digital labbook from 25 April 2025. | ||
{{Protokoll-Box}} | {{Protokoll-Box|titel=Immunoprecipitation (by MHE)|beschreibung=Immunoprecipitation is a molecular biology technique used for the specific isolation of proteins from complex protein mixtures such as cell lysates or tissue extracts. The method relies on the use of antibodies that specifically recognize and bind to a target protein of interest, effectively "fishing" it out of solution along with any associated proteins or complexes. | ||
The basic principle involves several key steps: first, an antibody specific to the target protein is added to the sample, where it forms an antigen-antibody complex. Next, Protein A or Protein G beads are introduced, which bind to the Fc region of the antibody, creating a larger complex that can be easily separated by centrifugation. After washing steps to remove non-specifically bound proteins, the target protein can be eluted and analyzed. | |||
Immunoprecipitation serves multiple important purposes in molecular biology research. It is commonly used for protein detection and validation, allowing researchers to confirm the presence and abundance of specific proteins in biological samples. One of its most valuable applications is in studying protein-protein interactions through co-immunoprecipitation (Co-IP), where proteins that associate with the target protein are co-purified and can be identified. The technique is also instrumental in analyzing post-translational modifications such as phosphorylation, ubiquitination, or acetylation, as these modifications are preserved during the gentle isolation process. | |||
Additionally, immunoprecipitation plays a crucial role in functional studies of protein complexes and signaling pathways, helping researchers understand how proteins work together in cellular processes. A specialized variant called Chromatin Immunoprecipitation (ChIP) is used to study DNA-protein interactions and gene regulation. The method offers high specificity due to antibody recognition, preserves native protein complexes, allows for quantitative analysis, and is compatible with downstream applications such as Western blotting, mass spectrometry, and proteomics analyses, making it an indispensable tool for studying protein biology and cellular signaling mechanisms.|materialien=Please refer to the realisation.|durchfuehrung=- THP-1 Cells (WT, PPM1F KO (clone D7), PPM1F KO + PPM1F WT (clone C9), PPM1F KO+ PPM1F D360A (clone A5)) were count and 3x106 cells were lysed using 1000 µl RIPA lysis buffer (10 µg/ml Aprotinin,10 µM Benzamidin, 5 µg/ml Leupepin, 1 µM PMSF, 10 nM pNPP, 2 µM cytochalasinD, cpd26, 1x Phosstop) | |||
- Cells were vortexted, sheared and sonificated for 7 sec, at 50% intensity | |||
- After 15 min centrifugation, at 14000 rpm and 4°C WCL were transferred into fresh tubes | |||
- 3 µg of antibodies were added to lysates (α-mouse hITGb2 (H52, #194) or α-mouse IgG (96/1) both supplied by AG Hauck) and incubated for 30 min at 4°C, while rotating | |||
- Then, 10 µl protein A/G beads were added for 45 min, at 4°C, while rotating | |||
- Suspension was centrifuged 2 min at 2600 rpm and 4°C | |||
- Beads were washed 2x using RIPA lysis buffer w/o proteases inhibitors | |||
- Afterwards beads were boiled for 5 min at 95 °C in 10 µl 2x SDS loading buffer | |||
- SDS-Page/WB (#765-766) | |||
o Blocking in blotto (1% BSA in TBS-T) for 1h at RT, while shaking | |||
o α-rabbit pT758 ITGb2 (1:1000 in Blotto) was added and incubation was performed o/N at 4°C, while shaking | |||
o the next day membrane was washed 3 h with TBS-T, 2nd AB (HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson, #17, 1:2000 in TBS-T) was added for 1h at RT, while shaking and evaluation in BioRad, chemiDOC was done using ECL supplemented with H2O2 | |||
o after evaluation, membrane was washed 3x in TBS-T for 10 min each step and blocked for 1h in Blotto, RT, while shaking | |||
o ITGb2 (H52), #194, 1:1000) was added and incubation was performed o/N at 4°C, while shaking | |||
o the next day membrane was washed 3 h with TBS-T, 2nd AB (HRP-conj. AffiniPure Goat anti-Mouse IgG (H+L), Jackson/Dianov., #11A, 1:2000 in TBS-T) was added for 1h at RT, while shaking and evaluation in BioRad, chemiDOC was done using ECL supplemented with H2O2 | |||
o evaluation was done by BioRAD (imageLab), Adobe photoshop, Adobe illustrator}} | |||
= Immunoprecipitation = | = Immunoprecipitation = | ||
Revision as of 12:49, 5 June 2025
This is an original excerpt from MHE's digital labbook from 25 April 2025.
🧪 Immunoprecipitation (by MHE)
Beschreibung
Immunoprecipitation is a molecular biology technique used for the specific isolation of proteins from complex protein mixtures such as cell lysates or tissue extracts. The method relies on the use of antibodies that specifically recognize and bind to a target protein of interest, effectively "fishing" it out of solution along with any associated proteins or complexes. The basic principle involves several key steps: first, an antibody specific to the target protein is added to the sample, where it forms an antigen-antibody complex. Next, Protein A or Protein G beads are introduced, which bind to the Fc region of the antibody, creating a larger complex that can be easily separated by centrifugation. After washing steps to remove non-specifically bound proteins, the target protein can be eluted and analyzed. Immunoprecipitation serves multiple important purposes in molecular biology research. It is commonly used for protein detection and validation, allowing researchers to confirm the presence and abundance of specific proteins in biological samples. One of its most valuable applications is in studying protein-protein interactions through co-immunoprecipitation (Co-IP), where proteins that associate with the target protein are co-purified and can be identified. The technique is also instrumental in analyzing post-translational modifications such as phosphorylation, ubiquitination, or acetylation, as these modifications are preserved during the gentle isolation process. Additionally, immunoprecipitation plays a crucial role in functional studies of protein complexes and signaling pathways, helping researchers understand how proteins work together in cellular processes. A specialized variant called Chromatin Immunoprecipitation (ChIP) is used to study DNA-protein interactions and gene regulation. The method offers high specificity due to antibody recognition, preserves native protein complexes, allows for quantitative analysis, and is compatible with downstream applications such as Western blotting, mass spectrometry, and proteomics analyses, making it an indispensable tool for studying protein biology and cellular signaling mechanisms.
Materialien
Please refer to the realisation.
Durchführung
- THP-1 Cells (WT, PPM1F KO (clone D7), PPM1F KO + PPM1F WT (clone C9), PPM1F KO+ PPM1F D360A (clone A5)) were count and 3x106 cells were lysed using 1000 µl RIPA lysis buffer (10 µg/ml Aprotinin,10 µM Benzamidin, 5 µg/ml Leupepin, 1 µM PMSF, 10 nM pNPP, 2 µM cytochalasinD, cpd26, 1x Phosstop)
- Cells were vortexted, sheared and sonificated for 7 sec, at 50% intensity - After 15 min centrifugation, at 14000 rpm and 4°C WCL were transferred into fresh tubes - 3 µg of antibodies were added to lysates (α-mouse hITGb2 (H52, #194) or α-mouse IgG (96/1) both supplied by AG Hauck) and incubated for 30 min at 4°C, while rotating - Then, 10 µl protein A/G beads were added for 45 min, at 4°C, while rotating - Suspension was centrifuged 2 min at 2600 rpm and 4°C - Beads were washed 2x using RIPA lysis buffer w/o proteases inhibitors - Afterwards beads were boiled for 5 min at 95 °C in 10 µl 2x SDS loading buffer - SDS-Page/WB (#765-766) o Blocking in blotto (1% BSA in TBS-T) for 1h at RT, while shaking o α-rabbit pT758 ITGb2 (1:1000 in Blotto) was added and incubation was performed o/N at 4°C, while shaking o the next day membrane was washed 3 h with TBS-T, 2nd AB (HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson, #17, 1:2000 in TBS-T) was added for 1h at RT, while shaking and evaluation in BioRad, chemiDOC was done using ECL supplemented with H2O2 o after evaluation, membrane was washed 3x in TBS-T for 10 min each step and blocked for 1h in Blotto, RT, while shaking o ITGb2 (H52), #194, 1:1000) was added and incubation was performed o/N at 4°C, while shaking o the next day membrane was washed 3 h with TBS-T, 2nd AB (HRP-conj. AffiniPure Goat anti-Mouse IgG (H+L), Jackson/Dianov., #11A, 1:2000 in TBS-T) was added for 1h at RT, while shaking and evaluation in BioRad, chemiDOC was done using ECL supplemented with H2O2 o evaluation was done by BioRAD (imageLab), Adobe photoshop, Adobe illustrator
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Immunoprecipitation
- THP-1 Cells (WT, PPM1F KO (clone D7), PPM1F KO + PPM1F WT (clone C9), PPM1F KO+ PPM1F D360A (clone A5)) were count and 3x106 cells were lysed using 1000 µl RIPA lysis buffer (10 µg/ml Aprotinin,10 µM Benzamidin, 5 µg/ml Leupepin, 1 µM PMSF, 10 nM pNPP, 2 µM cytochalasinD, cpd26, 1x Phosstop)
- Cells were vortexted, sheared and sonificated for 7 sec, at 50% intensity
- After 15 min centrifugation, at 14000 rpm and 4°C WCL were transferred into fresh tubes
- 3 µg of antibodies were added to lysates (α-mouse hITGb2 (H52, #194) or α-mouse IgG (96/1) both supplied by AG Hauck) and incubated for 30 min at 4°C, while rotating
- Then, 10 µl protein A/G beads were added for 45 min, at 4°C, while rotating
- Suspension was centrifuged 2 min at 2600 rpm and 4°C
- Beads were washed 2x using RIPA lysis buffer w/o proteases inhibitors
- Afterwards beads were boiled for 5 min at 95 °C in 10 µl 2x SDS loading buffer
- SDS-Page/WB (#765-766)
o Blocking in blotto (1% BSA in TBS-T) for 1h at RT, while shaking
o α-rabbit pT758 ITGb2 (1:1000 in Blotto) was added and incubation was performed o/N at 4°C, while shaking
o the next day membrane was washed 3 h with TBS-T, 2nd AB (HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson, #17, 1:2000 in TBS-T) was added for 1h at RT, while shaking and evaluation in BioRad, chemiDOC was done using ECL supplemented with H2O2
o after evaluation, membrane was washed 3x in TBS-T for 10 min each step and blocked for 1h in Blotto, RT, while shaking
o ITGb2 (H52), #194, 1:1000) was added and incubation was performed o/N at 4°C, while shaking
o the next day membrane was washed 3 h with TBS-T, 2nd AB (HRP-conj. AffiniPure Goat anti-Mouse IgG (H+L), Jackson/Dianov., #11A, 1:2000 in TBS-T) was added for 1h at RT, while shaking and evaluation in BioRad, chemiDOC was done using ECL supplemented with H2O2
o evaluation was done by BioRAD (imageLab), Adobe photoshop, Adobe illustrator