Adapted from Laemmli, U.K. (1970) Nature 227, 680-685. Yeast samples binding Notch were washed twice with 500 l of selection buffer, and incubated in 500 l of selection buffer supplemented with 100 nM of SA647 for 30 min at 4 C. 4% SDS 2x Laemmli buffer recipe. 2X Tris-Glycine SDS Sample buffer (Laemmli buffer) 4X LDS Sample Buffer; Electrophoresis running buffers. 4x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. The TGX gels retain Laemmli-like separation characteristics using the standard sample and Tris/Glycine running buffers. SAMPLE PREPARATION. A Bit of History. 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; 0.004% bromophenol blue; Sample preparation: Determine the protein concentration of each sample with a protein quantification assay (ie. (see page 681). Some samples may need to be reduced or denatured, this is achieved by boiling samples in buffer. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100C. Determine the protein concentration of each cell lysate. Troubleshooting tips. 4x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. 2X Laemmli loading buffer: Bromophenol blue (Product No. The Criterion TGX (Tris-Glycine eXtended) precast gels for PAGE are based on the long shelf life TGX formulation. 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; 0.004% bromophenol blue; Membrane stripping. The TGX gels retain Laemmli-like separation characteristics using the standard sample and Tris/Glycine running buffers. Introduction. Bradford assay). FAQ . Laemmli 2X buffer/loading buffer. Cleavage of structural proteins during the assembly of the head of bateriophage T4. The TGX gels retain Laemmli-like separation characteristics using the standard sample and Tris/Glycine running buffers. The proteins in the gel can be separated in as little as 20 min at 300 V while maintaining low temperatures. In parallel, a 5 l whole blood sample was added with 25 l HEPES buffer mixed with 10l anti-CD41 antibody and 10l IgG1K antibody and used as the negative control. Determine the protein concentration of each cell lysate. Add an equal volume of 2X Laemmli sample buffer to each sample. 10X Tris-Glycine SDS Running Buffer; SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL) Recipe for 2X buffer stock: 0.5 M Tris-HCl pH 6.8: Following incubation, the sample was exchanged by dialysis overnight using a 5 mL 500-1000Da MWCO dialysis cassette with 20 mM HEPES and 50 mM NaCl at pH 7.2. Introduction. 2X Laemmli Sample Buffer(2) abs9237. Roche NP-40 was used in cell lysis [50, 51]. Membrane stripping. 4x SDS-PAGE Sample Buffer 10x SDS-PAGE Running Buffer 125 mM TrisHCl, pH 6.8 1 M 5 ml 30.3 g Tris base Resuspend cells in 20 l of 2x sample buffer. Following incubation, the sample was exchanged by dialysis overnight using a 5 mL 500-1000Da MWCO dialysis cassette with 20 mM HEPES and 50 mM NaCl at pH 7.2. To retain native protein conformation and activity these gels can be run with sample and running buffer that do not contain SDS. The following is the composition of loading buffer required to prepare the samples for electrophoresis. NP-40. The following is the composition of loading buffer required to prepare the samples for electrophoresis. Loading buffer/Laemmli 2X buffer. Lysates can be aliquoted and stored at -20C for future use. 2x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. If necessary, heating the mix solution and then cool down the tube at room temperature. Western blot FAQ. SDS-PAGE Sample Buffer is specially formulated for protein sample preparation to based on the Laemmli Method of SDS-PAGE system. 2x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. Preparation of non-reducing sample A protein sample is mixed with the 2X SDS-PAGE sample buffer (1:1). Loading buffer/Laemmli 2X buffer. Detailed buffer and stock solution recipes for western blot, including TBS, medium stripping, RIPA buffer, Western blot sample prep. August 18, 2003 Edition Page 5 2x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. Lyse the cells by adding 75 - 100 L of modified 2x Laemmli's sample buffer (60 mM Tris-HCl pH 6.8, 12.5% glycerol, 2% SDS, 5% -mercaptoethanol (BME), and bromophenol blue; can be made up as a big batch, aliquoted, and stored at -20 C until use) or an equivalent sample buffer. We recently shut down crispr.mit.edu, but there are many other guide design tools available that we hope you will find helpful. Laemmli 2X buffer/loading buffer 4% SDS 10% 2-mercaptoethanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris-HCl Check the pH and adjust to 6.8 3. Determine how much protein to load (Recommended: 10-50 g/lane) and add an equal volume 2X Laemmli buffer. Nature, 227, 6805). For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. 3] Incubate tubes in boiling water for 5 min. Immunoprecipitated proteins were washed three times with lysis buffer, suspended in 2X Laemmli buffer and analyzed by immunoblotting. The proteins in the gel can be separated in as little as 20 min at 300 V while maintaining low temperatures. The 2X is to be mixed in 1:1 ratio with the sample. Electrophoresis: For standard denaturing electrophoresis use sample and running buffers containing SDS. Laemmli 2X buffer/loading buffer. 2X Laemmli Sample Buffer(2) abs9237. We recently shut down crispr.mit.edu, but there are many other guide design tools available that we hope you will find helpful. Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. Cells were collected 72 h after transfection and lysed in 1% NP-40 buffer (50 mM Tris-HCl, 150 mM NaCl and 1% NP-40, pH 7.6) with protease inhibitor (Roche, 4693159001). According to Linke , Laemmli sample buffer, 2D-DIGE experiments . In this article, well cover the components of Laemmli buffer, what they actually do, and end with a handy buffer recipe for your lab notebook. Lysates can be aliquoted and stored at -20C for future use. 10X Tris-Glycine SDS Running Buffer; SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL) Recipe for 2X buffer stock: 0.5 M Tris-HCl pH 6.8: To retain native protein conformation and activity these gels can be run with sample and running buffer that do not contain SDS. Determine how much protein to load (Recommended: 10-50 g/lane) and add an equal volume 2X Laemmli buffer. Add adequate ice-cold lysis buffer to make up all the lysates to the same volume. Transfer and staining. Add adequate ice-cold lysis buffer to make up all the lysates to the same volume. Immunoprecipitates were washed three times with lysis buffer supplemented with 500 mM or 200 mM NaCl (for FLAGS6K1 immunoprecipitation) and then eluted in 25 l 2 Laemmli. The Criterion TGX (Tris-Glycine eXtended) precast gels for PAGE are based on the long shelf life TGX formulation. In parallel, a 5 l whole blood sample was added with 25 l HEPES buffer mixed with 10l anti-CD41 antibody and 10l IgG1K antibody and used as the negative control. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. Designing primers for PCR based cloning: The basic PCR primers for molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site for cloning (usually 6-8bp) Hybridization Sequence: The region of the primer that binds to the sequence to be amplified Preparation of non-reducing sample A protein sample is mixed with the 2X SDS-PAGE sample buffer (1:1). 3] Incubate tubes in boiling water for 5 min. Western blot FAQ. 4x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. Troubleshooting tips. Add adequate ice-cold lysis buffer to make up all the lysates to the same volume. (see page 681). Directions for Use. Determine how much protein to load (Recommended: 10-50 g/lane) and add an equal volume 2X Laemmli buffer. Nature, 227, 6805). Adapted from Laemmli, U.K. (1970) Nature 227, 680-685. FAQ . The TGX gels retain Laemmli-like separation characteristics using the standard sample and Tris/Glycine running buffers. 2X Tris-Glycine SDS Sample buffer (Laemmli buffer) 4X LDS Sample Buffer; Electrophoresis running buffers. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. (see page 681). Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) *Not all percentages are available in every well type. Immunoprecipitated proteins were washed three times with lysis buffer, suspended in 2X Laemmli buffer and analyzed by immunoblotting. Laemmli 2X buffer/loading buffer 4% SDS 10% 2-mercaptoethanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris-HCl Check the pH and adjust to 6.8 3. The buffer is connected with the invention of SDS-PAGE during the quest for finding T4 phase proteins and got its name after the inventor Prof. Ulrich K. Laemmli [1].The composition has been discussed since the 70s and alternatives have been proposed. A protein sample is mixed with the 2X sample buffer (1:1) and heated in boiling water for 2-5 min. In most cases, the detergent concentration should be well about the CMC level (at least 2X the CMC) to ensure sufficient micelle concentration to solubilize the membrane proteins. Immunoprecipitates were washed three times with lysis buffer supplemented with 500 mM or 200 mM NaCl (for FLAGS6K1 immunoprecipitation) and then eluted in 25 l 2 Laemmli. Yeast samples binding Notch were washed twice with 500 l of selection buffer, and incubated in 500 l of selection buffer supplemented with 100 nM of SA647 for 30 min at 4 C. Detailed buffer and stock solution recipes for western blot, including TBS, medium stripping, RIPA buffer, Western blot sample prep. Introduction. Troubleshooting tips. Bradford assay). Mix samples with Laemmli loading buffer. WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) *Not all percentages are available in every well type. The buffer is connected with the invention of SDS-PAGE during the quest for finding T4 phase proteins and got its name after the inventor Prof. Ulrich K. Laemmli [1].The composition has been discussed since the 70s and alternatives have been proposed. 4% SDS 3] Incubate tubes in boiling water for 5 min. Electrophoresis: The supernatant was then incubated with 1 g of anti- FLAG antibody for 2 hours at 4C, followed by addition of protein G Sepharose beads (P3296, Sigma-Aldrich) for 1 hour at 4C. Designing primers for PCR based cloning: The basic PCR primers for molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site for cloning (usually 6-8bp) Hybridization Sequence: The region of the primer that binds to the sequence to be amplified Lyse the cells by adding 75 - 100 L of modified 2x Laemmli's sample buffer (60 mM Tris-HCl pH 6.8, 12.5% glycerol, 2% SDS, 5% -mercaptoethanol (BME), and bromophenol blue; can be made up as a big batch, aliquoted, and stored at -20 C until use) or an equivalent sample buffer. The TGX gels retain Laemmli-like separation characteristics using the standard sample and Tris/Glycine running buffers. Preparation of non-reducing sample A protein sample is mixed with the 2X SDS-PAGE sample buffer (1:1). For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. The proteins in the gel can be separated in as little as 20 min at 300 V while maintaining low temperatures. Lysates can be aliquoted and stored at Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. Yeast samples binding Notch were washed twice with 500 l of selection buffer, and incubated in 500 l of selection buffer supplemented with 100 nM of SA647 for 30 min at 4 C. Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. 4x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. The proteins in the gel can be separated in as little as 20 min at 300 V while maintaining low temperatures. August 18, 2003 Edition Page 5 Lysates can be aliquoted and stored at -20C for future use. If necessary, heating the mix solution and then cool down the tube at room temperature. 2x Laemmli buffer recipe. Non-reducing 4X LDS sample buffer was added to sample lysate, followed by boiling at 100 C for 10 min and samples were loaded on a 3% stacking and 8% separating SDS-PAGE. It can also be made at 4X and 6X strength to minimize dilution of the samples. Cleavage of structural proteins during the assembly of the head of bateriophage T4. Mix samples with Laemmli loading buffer. 2X Laemmli loading buffer: Bromophenol blue (Product No. Laemmli buffer takes its name from Professor Ulrich K. Laemmli, who Cells were collected 72 h after transfection and lysed in 1% NP-40 buffer (50 mM Tris-HCl, 150 mM NaCl and 1% NP-40, pH 7.6) with protease inhibitor (Roche, 4693159001). The supernatant was then incubated with 1 g of anti- FLAG antibody for 2 hours at 4C, followed by addition of protein G Sepharose beads (P3296, Sigma-Aldrich) for 1 hour at 4C. We recently shut down crispr.mit.edu, but there are many other guide design tools available that we hope you will find helpful. The 2-mercaptoethanol reduces the intra and inter-molecular disulfide bonds. Roche NP-40 was used in cell lysis [50, 51]. A Bit of History. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100C. The Laemmle sample buffer is used for the better isolation of proteins in SDS-PAGE gel electrophoresis. The Laemmle sample buffer is used for the better isolation of proteins in SDS-PAGE gel electrophoresis. Bradford assay). The standard loading buffer is called 2X Laemmli buffer (Laemmli UK, 1970. In this article, well cover the components of Laemmli buffer, what they actually do, and end with a handy buffer recipe for your lab notebook. 4% SDS Directions for Use. Some samples may need to be reduced or denatured, this is achieved by boiling samples in buffer. Following incubation, the sample was exchanged by dialysis overnight using a 5 mL 500-1000Da MWCO dialysis cassette with 20 mM HEPES and 50 mM NaCl at pH 7.2. Mix samples with Laemmli loading buffer. To reduce and denature your samples, boil each cell lysate in sample buffer at 100C for 5 min. In most cases, the detergent concentration should be well about the CMC level (at least 2X the CMC) to ensure sufficient micelle concentration to solubilize the membrane proteins. Use 2x Laemmli Sample Buffer for preparation of samples for SDS PAGE. Sample preparation: Determine the protein concentration of each sample with a protein quantification assay (ie. 2X Laemmli Sample Buffer(2) abs9237. The Criterion TGX (Tris-Glycine eXtended) precast gels for PAGE are based on the long shelf life TGX formulation. In parallel, a 5 l whole blood sample was added with 25 l HEPES buffer mixed with 10l anti-CD41 antibody and 10l IgG1K antibody and used as the negative control. Laemmli 2X buffer/loading buffer. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100C. The TGX gels retain Laemmli-like separation characteristics using the standard sample and Tris/Glycine running buffers. Laemmli sample buffer is especially formulated for protein sample preparation to be used in the Laemmli SDS-PAGE system. Reduce and denature the samples by boiling NP-40. Immunoprecipitates were washed three times with lysis buffer supplemented with 500 mM or 200 mM NaCl (for FLAGS6K1 immunoprecipitation) and then eluted in 25 l 2 Laemmli. Cells were collected 72 h after transfection and lysed in 1% NP-40 buffer (50 mM Tris-HCl, 150 mM NaCl and 1% NP-40, pH 7.6) with protease inhibitor (Roche, 4693159001). Laemmli buffer takes its name from Professor Ulrich K. Laemmli, who Reduce and denature the samples by boiling Thank you to the thousands of users who visited our guide design tool over the past five years. The supernatant was then incubated with 1 g of anti- FLAG antibody for 2 hours at 4C, followed by addition of protein G Sepharose beads (P3296, Sigma-Aldrich) for 1 hour at 4C. Lyse the cells by adding 75 - 100 L of modified 2x Laemmli's sample buffer (60 mM Tris-HCl pH 6.8, 12.5% glycerol, 2% SDS, 5% -mercaptoethanol (BME), and bromophenol blue; can be made up as a big batch, aliquoted, and stored at -20 C until use) or an equivalent sample buffer. Directions for Use. Add an equal volume of 2X Laemmli sample buffer to each sample. Adapted from Laemmli, U.K. (1970) Nature 227, 680-685. The Criterion TGX (Tris-Glycine eXtended) precast gels for PAGE are based on the long shelf life TGX formulation. 2x Laemmli buffer recipe. Determine the protein concentration of each cell lysate. Loading buffer/Laemmli 2X buffer. A protein sample is mixed with the 2X sample buffer (1:1) and heated in boiling water for 2-5 min. In most cases, the detergent concentration should be well about the CMC level (at least 2X the CMC) to ensure sufficient micelle concentration to solubilize the membrane proteins. Designing primers for PCR based cloning: The basic PCR primers for molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site for cloning (usually 6-8bp) Hybridization Sequence: The region of the primer that binds to the sequence to be amplified The Criterion TGX (Tris-Glycine eXtended) precast gels for PAGE are based on the long shelf life TGX formulation. NP-40. The standard loading buffer is called 2X Laemmli buffer (Laemmli UK, 1970. Laemmli sample buffer is especially formulated for protein sample preparation to be used in the Laemmli SDS-PAGE system. August 18, 2003 Edition Page 5 SAMPLE PREPARATION. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. 2X Laemmli loading buffer: Bromophenol blue (Product No. Laemmli sample buffer is especially formulated for protein sample preparation to be used in the Laemmli SDS-PAGE system. For standard denaturing electrophoresis use sample and running buffers containing SDS. The Laemmle sample buffer is used for the better isolation of proteins in SDS-PAGE gel electrophoresis. Transfer and staining. 10X Tris-Glycine SDS Running Buffer; SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL) Recipe for 2X buffer stock: 0.5 M Tris-HCl pH 6.8: Membrane stripping. The Criterion TGX (Tris-Glycine eXtended) precast gels for PAGE are based on the long shelf life TGX formulation. The 2-mercaptoethanol reduces the intra and inter-molecular disulfide bonds. Roche NP-40 was used in cell lysis [50, 51]. Electrophoresis: In this article, well cover the components of Laemmli buffer, what they actually do, and end with a handy buffer recipe for your lab notebook. To reduce and denature your samples, boil each cell lysate in sample buffer at 100C for 5 min. Immunoprecipitated proteins were washed three times with lysis buffer, suspended in 2X Laemmli buffer and analyzed by immunoblotting. Add an equal volume of 2X Laemmli sample buffer to each sample. 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; 0.004% bromophenol blue; 0.125 M Tris-HCl; To reduce and denature your samples, boil each cell lysate in sample buffer at 100C for 5 min. It can also be made at 4X and 6X strength to minimize dilution of the samples. If necessary, heating the mix solution and then cool down the tube at room temperature. 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; 0.004% bromophenol blue; 0.125 M Tris-HCl; To reduce and denature your samples, boil each cell lysate in sample buffer at 100C for 5 min. 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; 0.004% bromophenol blue; Laemmli buffer takes its name from Professor Ulrich K. Laemmli, who Transfer and staining. A protein sample is mixed with the 2X sample buffer (1:1) and heated in boiling water for 2-5 min. SAMPLE PREPARATION. Lysates can be aliquoted and stored at FAQ . 4x SDS-PAGE Sample Buffer 10x SDS-PAGE Running Buffer 125 mM TrisHCl, pH 6.8 1 M 5 ml 30.3 g Tris base Resuspend cells in 20 l of 2x sample buffer. 2X Tris-Glycine SDS Sample buffer (Laemmli buffer) 4X LDS Sample Buffer; Electrophoresis running buffers. Sample preparation: Determine the protein concentration of each sample with a protein quantification assay (ie. Detailed buffer and stock solution recipes for western blot, including TBS, medium stripping, RIPA buffer, Western blot sample prep. Use 2x Laemmli Sample Buffer for preparation of samples for SDS PAGE. SDS-PAGE Sample Buffer is specially formulated for protein sample preparation to based on the Laemmli Method of SDS-PAGE system. 4x Laemmli Sample Buffer can be used with the following Mini-PROTEAN and midi Criterion Precast Protein Gels. The proteins in the gel can be separated in as little as 20 min at 300 V while maintaining low temperatures. To reduce and denature your samples, boil each cell lysate in sample buffer at 100C for 5 min. Cleavage of structural proteins during the assembly of the head of bateriophage T4. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. Laemmli 2X buffer/loading buffer 4% SDS 10% 2-mercaptoethanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris-HCl Check the pH and adjust to 6.8 3. Thank you to the thousands of users who visited our guide design tool over the past five years. For standard denaturing electrophoresis use sample and running buffers containing SDS. 4x SDS-PAGE Sample Buffer 10x SDS-PAGE Running Buffer 125 mM TrisHCl, pH 6.8 1 M 5 ml 30.3 g Tris base Resuspend cells in 20 l of 2x sample buffer. Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. The 2X is to be mixed in 1:1 ratio with the sample. A Bit of History. Lysates can be aliquoted and stored at To retain native protein conformation and activity these gels can be run with sample and running buffer that do not contain SDS. Reduce and denature the samples by boiling The 2X is to be mixed in 1:1 ratio with the sample. The buffer is connected with the invention of SDS-PAGE during the quest for finding T4 phase proteins and got its name after the inventor Prof. Ulrich K. Laemmli [1].The composition has been discussed since the 70s and alternatives have been proposed. Western blot FAQ. The following is the composition of loading buffer required to prepare the samples for electrophoresis. Thank you to the thousands of users who visited our guide design tool over the past five years. Non-reducing 4X LDS sample buffer was added to sample lysate, followed by boiling at 100 C for 10 min and samples were loaded on a 3% stacking and 8% separating SDS-PAGE. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample. Some samples may need to be reduced or denatured, this is achieved by boiling samples in buffer. It can also be made at 4X and 6X strength to minimize dilution of the samples. Non-reducing 4X LDS sample buffer was added to sample lysate, followed by boiling at 100 C for 10 min and samples were loaded on a 3% stacking and 8% separating SDS-PAGE. Use 2x Laemmli Sample Buffer for preparation of samples for SDS PAGE. WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) *Not all percentages are available in every well type. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample.

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