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Authors: Nus Igem

Proper citation: Nus Igem 2019. Preparation of Chemically Competent Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.7pnhmme Copy   

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Authors: Karina Conkrite
Group: Diskin Lab-CHOP
Summary: From growth to isolation and lysis, to quantification, and running the gel, transfer, blotting... the whole bit.

Proper citation: Karina Conkrite 2019. Western Blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.6rmhd46 Copy   

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Authors: Audrey Lapinaite, Sam Sternberg, Brett Staahl
Group: The Center for Genome Editing and Recording
Summary: This is a protocol from the Doudna Lab for His10-MBP-Cas9 purification. 

Proper citation: Audrey Lapinaite, Sam Sternberg, Brett Staahl 2018. His10-MBP-Cas9 purification. protocols.io https://dx.doi.org/10.17504/protocols.io.ssseeee Copy   

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Authors: Hongfang Zhu

Proper citation: Hongfang Zhu 2017. Assay of anthocyanin biosynthetic enzyme activity. protocols.io https://dx.doi.org/10.17504/protocols.io.h2mb8c6 Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol used to dissociate adult (8-10 wk) mouse spleen into single cells. Attained >95% viability, a variety of cell sizes, and ~10 million cells from 12 mg tissue.

Proper citation: Andrew Potter 2018. Adult Mouse Spleen Dissociation (On ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q5cdy2w Copy   

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Authors: LGC Biosearch Technologies
Summary: Stellaris RNA FISH protocol to simultaneously label adherent cells with IF and RNA FISH.

Proper citation: LGC Biosearch Technologies 2016. Stellaris® RNA FISH Simultaneous IF + FISH in Adherent Cells Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ek8bczw Copy   

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Authors: Mohammad Z. Islam, Andrei Fokine, Marthandan Mahalingam, Zhihong Zhang, Carmela Garcia-Doval, Mark J. van Raaij, Michael G. Rossmann, and Venigalla B. Rao
Summary: Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.

Proper citation: Mohammad Z. Islam, Andrei Fokine, Marthandan Mahalingam, Zhihong Zhang, Carmela Garcia-Doval, Mark J. van Raaij, Michael G. Rossmann, and Venigalla B. Rao 2019. Formation of gp37 LTF needle trimers-LPS complex. protocols.io https://dx.doi.org/10.17504/protocols.io.77nhrme Copy   

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Authors: Alba Balletbó
Group: iGEM Wageningen 2019
Summary: The following protocol is suitable for PCR clean-up as well as DNA concentration and removal of salts, enzymes, etc. from enzymatic reactions (SDS

Proper citation: Alba Balletbó 2019. DNA Purification from a PCR Product (Protocol for NucleoSpin® PCR clean-up Gel Extraction Kit). protocols.io https://dx.doi.org/10.17504/protocols.io.7jahkie Copy   

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Authors: Hiraku Miyagi, Michio Hiroshima, Yasushi Sako
Summary: Immunofluorescence microscopy is used to determine molecular localization and to measure fluorescence intensity of activity in cultured cells. Reagents for fixation and/or permeabilization must be suitable for the molecule of interest. We developed a procedure to immunostain phosphatidylinositol biphosphate (PIP2) and phosphorylated Extracellular signal-Regulated Kinase (ERK) simultaneously. PIP2 is a phospholipid component of cell membranes. ERK is a mitogen-activated protein kinase (MAPK) that localized in the cytoplasm and nucleus after phosphorylation triggered by ligand activation. Different compounds are used to permeabilize cells in order to stain for these two molecules. Digitonin is used for PIP2 and Triton X-100 is used for phosphorylated ERK. However, since Triton X-100 can solubilize cell membrane lipids, it is not suitable for PIP2 labeling. In order to confer resistance against Triton X-100, we devised a method to fix anti-PIP2 antibody on the cell membrane antigen before permeabilization with Triton X-100. We modified a conventional immunostaining method to include secondary fixation and permeabilization after anti-PIP2 antibody labeling. The secondary fixation probably immobilizes antibodies bound to PIP2 on the cell membrane, perhaps by cross-linking them to surrounding protein molecules. Whatever the mechanism, this fixation enables PIP2-antibody complexes to remain on the membrane, despite treatment with Triton X-100, which is used to visualize phosphorylated ERK. Since PIP2 and ERK are major cell signaling molecules in various cellular phenomena, simultaneous detection and visualization of their phosphorylation states by this method should be useful for many studies.

Proper citation: Hiraku Miyagi, Michio Hiroshima, Yasushi Sako 2019. Double immunostaining for PIP2 and phosphorylated ERK. protocols.io https://dx.doi.org/10.17504/protocols.io.9gxh3xn Copy   

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Authors: Larissa Andrade, João Pedro Santos, María Cristina Vidal Pessolani
Summary: Goal:This document has the objective of standardizing the protocol for Western blot. This technique allows the detection of specific proteins separated on polyacrylamide gel and transferred to a membrane, using antibodies.General considerations:Before the experiment, check whether all the necessary solutions are already prepared, i.e., transfer buffer, TBS, and TBST (recipes are below).This protocol takes into account that the protein extracts for evaluation were previously separated through SDS-PAGE. After the gel electrophoresis, handle the gel gently to prevent damage.* It is recommended to use a pre-stained molecular weight standard on SDS-PAGE, in order to follow the protein separation during the electrophoresis and to check whether the transfer has worked.**Do not stain the SDS-PAGE gel. Right after the electrophoresis transfer the proteins to the nitrocellulose or PVDF membrane as described below.This protocol has two main steps. The initial step is the protein transfer from a polyacrylamide gel onto a membrane. The following step is the incubation of the membrane with antibodies and chemiluminescent development. In every step, it is necessary to wear gloves to prevent leaving any finger marks or any sort of spot on the membrane. Experimental procedures: 1. Buffer recipes: - Transfer buffer 10x: Add 18.9 g Tris and 90 g glycine to a beaker containing approximately 700mL Milli-Q water. Homogenize with a magnetic bar. Transfer the solution to a 1 L graduated cylinder and fill it with Milli-Q water up until 1L. Store the solution at room temperature labeling as “Transfer Buffer 10x”. - Transfer Buffer 1x (must be prepared fresh and should be reused only once): Add to a graduated cylinder 100 mL of Transfer Buffer 10x, 200 mL ethanol 100% and fill it with Milli-Q water up until 1 L.  Seal it with Parafilm and homogenize by inversion. - Tris Buffered Saline (TBS) 10x: Add 24.2 g Tris and 87.6 g NaCl to a beaker containing approximately 700 mL Milli-Q water. Homogenize with a magnetic bar and adjust the pH to 7.4 adding the necessary amount of hydrochloric acid (HCl) according to the pH Meter. After adjusting the pH, transfer to a graduated cylinder and fill it with Milli-Q water up until 1 L. Seal it with Parafilm and homogenize by inversion. Store the solution at room temperature labeling as “TBS 10x”. - TBS 1x: Add 100 mL TBS 10x to a graduated cylinder, fill it with Milli-Q water up until 1L and homogenize through inversion sealing with Parafilm. - TBS-Tween 0.1% (TBS-T): Add 100 mL TBS 10x and 1 mL Tween 20 to a graduated cylinder, fill it with Milli-Q water up until 1L, seal it with Parafilm and homogenize by inversion. Transfer:- Right after the gel electrophoresis, add circa 100 mL of transfer buffer 1x to a recipient and insert the gel into this solution.- Pick up a transfer tank. Put the transfer tank into a Styrofoam and fill it with ice.- Pick up a transfer cassette, 2 sponges, 6 filter papers, and cut a piece of nitrocellulose or PVDF membrane with the same size as the polyacrylamide gel.      The nitrocellulose demands use of methanol on the transfer buffer, which might reduce the pore size and result in precipitation of high molecular weight proteins. On the other hand, PVDF membrane is recommended for high molecular weight proteins, linking to the membrane through hydrophobic and dipolar interactions, therefore with better linkage capacity. Before transferring, it is necessary to moist the membrane with methanol or ethanol to activate it (immersion for 30 seconds). The membrane must be cut with the size of the gel, in order to avoid waste of it, owing to its high cost. Make a small cut on the corner where the molecular weight marker is located to mark the sample loading orientation. - Add transfer buffer to a big recipient, glass or plastic, and use it to assemble the transfer “sandwich” as follows:Put the transfer cassette with the red (or white) side turned down and open the cassette. Add in the following order: 1 sponge, 3 filter papers, the membrane (with the exact size of the gel), the gel, 3 filter papers, 1 sponge. Close the cassette.* Remove any bubbles between the gel and the membrane since it prevents the proteins transfer. As such, each layer must be pressed gently along its addition to the cassette in order to redraw the air.The transfer must be set up as shown below: *Attention: The gel must be always located on the black side of the cassette (negative pole) and the membrane on the red or white side (positive pole). The proteins will be transferred from the negative (gel) to the positive (membrane) pole, so a lot of care must be taken when setting up the transfer “sandwich” on the right position. - The cassette must be placed on the transfer tank, minding that the black side of the cassette be placed closer to the black electrode (negative pole) and the red or white side of the cassette closer to the red electrode (positive pole). - Fill the tank with Transfer Buffer 1x. There must be enough buffer to cover up the entire cassette where the gel is placed. On the transfer tank there is a mark defining the specific height of buffer to be filled. * It is possible to transfer up to 4 gels simultaneously. - The lid of the transfer tank has a black and a red wire. Close the tank positioning the lid so that the black electrode connects to the black wire (negative pole) and the red or white electrode connects to the red wire (positive pole). - Connect the wires to the power supply, again placing the black wire to the black slot and the red wire to the red slot. Turn on the power supply and adjust it to 100V and 400mA for 1h. Alternatively, it might be transferred overnight on 10V (in this case, it is better to set up the transfer system into a fridge or a cold chamber, because the ice will melt before finishing the transfer). When turning on the power supply, it is necessary to observe whether the electric current is properly transmitted by checking the Amperage and the production of bubbles into the tank.  - After the transfer, redraw carefully the cassette from the transfer tank. In the case of using a prestained molecular weight standard, this step must take into account whether the bands of the prestained standard have been transferred from the gel to the membrane. Transfer the membrane to a plastic or glass recipient with lid. - Stain membrane with 0.1% Amido Black Solution and leave the solution until the bands appear, homogenizing gently. This staining allows the protein bands (samples) to be seen in the membrane, i.e., whether the proteins have been transferred. With aid of tweezers, put the staining solution back to its recipient and mark the position of the molecular weight standard bands with a pencil (in the case of non prestained). Take a picture of the membrane, and then wash it with distilled water three times, 10 minutes each, homogenizing gently, to destain the membrane. Blocking, incubation with antibodies and development:* It is recommended to use a pre-stained molecular weight standard on SDS-PAGE, in order to follow the protein separation during the electrophoresis and to check whether the transfer has worked.**Do not stain the SDS-PAGE gel. Right after the electrophoresis transfer the proteins to the nitrocellulose or PVDF membrane as described below.This protocol has two main steps. The initial step is the protein transfer from a polyacrylamide gel onto a membrane. The following step is the incubation of the membrane with antibodies and chemiluminescent development. In every step, it is necessary to wear gloves to prevent leaving any finger marks or any sort of spot on the membrane. Experimental procedures: 1. Buffer recipes: - Transfer buffer 10x: Add 18.9 g Tris and 90 g glycine to a beaker containing approximately 700mL Milli-Q water. Homogenize with a magnetic bar. Transfer the solution to a 1 L graduated cylinder and fill it with Milli-Q water up until 1L. Store the solution at room temperature labeling as “Transfer Buffer 10x”. - Transfer Buffer 1x (must be prepared fresh and should be reused only once): Add to a graduated cylinder 100 mL of Transfer Buffer 10x, 200 mL ethanol 100% and fill it with Milli-Q water up until 1 L.  Seal it with Parafilm and homogenize by inversion. - Tris Buffered Saline (TBS) 10x: Add 24.2 g Tris and 87.6 g NaCl to a beaker containing approximately 700 mL Milli-Q water. Homogenize with a magnetic bar and adjust the pH to 7.4 adding the necessary amount of hydrochloric acid (HCl) according to the pH Meter. After adjusting the pH, transfer to a graduated cylinder and fill it with Milli-Q water up until 1 L. Seal it with Parafilm and homogenize by inversion. Store the solution at room temperature labeling as “TBS 10x”. - TBS 1x: Add 100 mL TBS 10x to a graduated cylinder, fill it with Milli-Q water up until 1L and homogenize through inversion sealing with Parafilm. - TBS-Tween 0.1% (TBS-T): Add 100 mL TBS 10x and 1 mL Tween 20 to a graduated cylinder, fill it with Milli-Q water up until 1L, seal it with Parafilm and homogenize by inversion. Transfer:- Right after the gel electrophoresis, add circa 100 mL of transfer buffer 1x to a recipient and insert the gel into this solution.- Pick up a transfer tank. Put the transfer tank into a Styrofoam and fill it with ice.- Pick up a transfer cassette, 2 sponges, 6 filter papers, and cut a piece of nitrocellulose or PVDF membrane with the same size as the polyacrylamide gel.      The nitrocellulose demands use of methanol on the transfer buffer, which might reduce the pore size and result in precipitation of high molecular weight proteins. On the other hand, PVDF membrane is recommended for high molecular weight proteins, linking to the membrane through hydrophobic and dipolar interactions, therefore with better linkage capacity. Before transferring, it is necessary to moist the membrane with methanol or ethanol to activate it (immersion for 30 seconds). The membrane must be cut with the size of the gel, in order to avoid waste of it, owing to its high cost. Make a small cut on the corner where the molecular weight marker is located to mark the sample loading orientation. - Add transfer buffer to a big recipient, glass or plastic, and use it to assemble the transfer “sandwich” as follows:Put the transfer cassette with the red (or white) side turned down and open the cassette. Add in the following order: 1 sponge, 3 filter papers, the membrane (with the exact size of the gel), the gel, 3 filter papers, 1 sponge. Close the cassette.* Remove any bubbles between the gel and the membrane since it prevents the proteins transfer. As such, each layer must be pressed gently along its addition to the cassette in order to redraw the air.The transfer must be set up as shown below: *Attention: The gel must be always located on the black side of the cassette (negative pole) and the membrane on the red or white side (positive pole). The proteins will be transferred from the negative (gel) to the positive (membrane) pole, so a lot of care must be taken when setting up the transfer “sandwich” on the right position. - The cassette must be placed on the transfer tank, minding that the black side of the cassette be placed closer to the black electrode (negative pole) and the red or white side of the cassette closer to the red electrode (positive pole). - Fill the tank with Transfer Buffer 1x. There must be enough buffer to cover up the entire cassette where the gel is placed. On the transfer tank there is a mark defining the specific height of buffer to be filled. * It is possible to transfer up to 4 gels simultaneously. - The lid of the transfer tank has a black and a red wire. Close the tank positioning the lid so that the black electrode connects to the black wire (negative pole) and the red or white electrode connects to the red wire (positive pole). - Connect the wires to the power supply, again placing the black wire to the black slot and the red wire to the red slot. Turn on the power supply and adjust it to 100V and 400mA for 1h. Alternatively, it might be transferred overnight on 10V (in this case, it is better to set up the transfer system into a fridge or a cold chamber, because the ice will melt before finishing the transfer). When turning on the power supply, it is necessary to observe whether the electric current is properly transmitted by checking the Amperage and the production of bubbles into the tank.  - After the transfer, redraw carefully the cassette from the transfer tank. In the case of using a prestained molecular weight standard, this step must take into account whether the bands of the prestained standard have been transferred from the gel to the membrane. Transfer the membrane to a plastic or glass recipient with lid. - Stain membrane with 0.1% Amido Black Solution and leave the solution until the bands appear, homogenizing gently. This staining allows the protein bands (samples) to be seen in the membrane, i.e., whether the proteins have been transferred. With aid of tweezers, put the staining solution back to its recipient and mark the position of the molecular weight standard bands with a pencil (in the case of non prestained). Take a picture of the membrane, and then wash it with distilled water three times, 10 minutes each, homogenizing gently, to destain the membrane. Blocking, incubation with antibodies and development:Experimental procedures:1. Buffer recipes:- Transfer buffer 10x: Add 18.9 g Tris and 90 g glycine to a beaker containing approximately 700mL Milli-Q water. Homogenize with a magnetic bar. Transfer the solution to a 1 L graduated cylinder and fill it with Milli-Q water up until 1L. Store the solution at room temperature labeling as “Transfer Buffer 10x”.- Transfer Buffer 1x (must be prepared fresh and should be reused only once): Add to a graduated cylinder 100 mL of Transfer Buffer 10x, 200 mL ethanol 100% and fill it with Milli-Q water up until 1 L.  Seal it with Parafilm and homogenize by inversion.- Tris Buffered Saline (TBS) 10x: Add 24.2 g Tris and 87.6 g NaCl to a beaker containing approximately 700 mL Milli-Q water. Homogenize with a magnetic bar and adjust the pH to 7.4 adding the necessary amount of hydrochloric acid (HCl) according to the pH Meter. After adjusting the pH, transfer to a graduated cylinder and fill it with Milli-Q water up until 1 L. Seal it with Parafilm and homogenize by inversion. Store the solution at room temperature labeling as “TBS 10x”.- TBS 1x: Add 100 mL TBS 10x to a graduated cylinder, fill it with Milli-Q water up until 1L and homogenize through inversion sealing with Parafilm.- TBS-Tween 0.1% (TBS-T): Add 100 mL TBS 10x and 1 mL Tween 20 to a graduated cylinder, fill it with Milli-Q water up until 1L, seal it with Parafilm and homogenize by inversion.Transfer:- Right after the gel electrophoresis, add circa 100 mL of transfer buffer 1x to a recipient and insert the gel into this solution.- Pick up a transfer tank. Put the transfer tank into a Styrofoam and fill it with ice.- Pick up a transfer cassette, 2 sponges, 6 filter papers, and cut a piece of nitrocellulose or PVDF membrane with the same size as the polyacrylamide gel.      The nitrocellulose demands use of methanol on the transfer buffer, which might reduce the pore size and result in precipitation of high molecular weight proteins. On the other hand, PVDF membrane is recommended for high molecular weight proteins, linking to the membrane through hydrophobic and dipolar interactions, therefore with better linkage capacity. Before transferring, it is necessary to moist the membrane with methanol or ethanol to activate it (immersion for 30 seconds). The membrane must be cut with the size of the gel, in order to avoid waste of it, owing to its high cost. Make a small cut on the corner where the molecular weight marker is located to mark the sample loading orientation. - Add transfer buffer to a big recipient, glass or plastic, and use it to assemble the transfer “sandwich” as follows:Put the transfer cassette with the red (or white) side turned down and open the cassette. Add in the following order: 1 sponge, 3 filter papers, the membrane (with the exact size of the gel), the gel, 3 filter papers, 1 sponge. Close the cassette.* Remove any bubbles between the gel and the membrane since it prevents the proteins transfer. As such, each layer must be pressed gently along its addition to the cassette in order to redraw the air.The transfer must be set up as shown below: *Attention: The gel must be always located on the black side of the cassette (negative pole) and the membrane on the red or white side (positive pole). The proteins will be transferred from the negative (gel) to the positive (membrane) pole, so a lot of care must be taken when setting up the transfer “sandwich” on the right position. - The cassette must be placed on the transfer tank, minding that the black side of the cassette be placed closer to the black electrode (negative pole) and the red or white side of the cassette closer to the red electrode (positive pole). - Fill the tank with Transfer Buffer 1x. There must be enough buffer to cover up the entire cassette where the gel is placed. On the transfer tank there is a mark defining the specific height of buffer to be filled. * It is possible to transfer up to 4 gels simultaneously. - The lid of the transfer tank has a black and a red wire. Close the tank positioning the lid so that the black electrode connects to the black wire (negative pole) and the red or white electrode connects to the red wire (positive pole). - Connect the wires to the power supply, again placing the black wire to the black slot and the red wire to the red slot. Turn on the power supply and adjust it to 100V and 400mA for 1h. Alternatively, it might be transferred overnight on 10V (in this case, it is better to set up the transfer system into a fridge or a cold chamber, because the ice will melt before finishing the transfer). When turning on the power supply, it is necessary to observe whether the electric current is properly transmitted by checking the Amperage and the production of bubbles into the tank.  - After the transfer, redraw carefully the cassette from the transfer tank. In the case of using a prestained molecular weight standard, this step must take into account whether the bands of the prestained standard have been transferred from the gel to the membrane. Transfer the membrane to a plastic or glass recipient with lid. - Stain membrane with 0.1% Amido Black Solution and leave the solution until the bands appear, homogenizing gently. This staining allows the protein bands (samples) to be seen in the membrane, i.e., whether the proteins have been transferred. With aid of tweezers, put the staining solution back to its recipient and mark the position of the molecular weight standard bands with a pencil (in the case of non prestained). Take a picture of the membrane, and then wash it with distilled water three times, 10 minutes each, homogenizing gently, to destain the membrane. Blocking, incubation with antibodies and development:- Right after the gel electrophoresis, add circa 100 mL of transfer buffer 1x to a recipient and insert the gel into this solution.- Pick up a transfer tank. Put the transfer tank into a Styrofoam and fill it with ice.- Pick up a transfer cassette, 2 sponges, 6 filter papers, and cut a piece of nitrocellulose or PVDF membrane with the same size as the polyacrylamide gel.     The nitrocellulose demands use of methanol on the transfer buffer, which might reduce the pore size and result in precipitation of high molecular weight proteins. On the other hand, PVDF membrane is recommended for high molecular weight proteins, linking to the membrane through hydrophobic and dipolar interactions, therefore with better linkage capacity. Before transferring, it is necessary to moist the membrane with methanol or ethanol to activate it (immersion for 30 seconds). The membrane must be cut with the size of the gel, in order to avoid waste of it, owing to its high cost. Make a small cut on the corner where the molecular weight marker is located to mark the sample loading orientation.- Add transfer buffer to a big recipient, glass or plastic, and use it to assemble the transfer “sandwich” as follows:Put the transfer cassette with the red (or white) side turned down and open the cassette. Add in the following order: 1 sponge, 3 filter papers, the membrane (with the exact size of the gel), the gel, 3 filter papers, 1 sponge. Close the cassette.* Remove any bubbles between the gel and the membrane since it prevents the proteins transfer. As such, each layer must be pressed gently along its addition to the cassette in order to redraw the air.The transfer must be set up as shown below:*Attention: The gel must be always located on the black side of the cassette (negative pole) and the membrane on the red or white side (positive pole). The proteins will be transferred from the negative (gel) to the positive (membrane) pole, so a lot of care must be taken when setting up the transfer “sandwich” on the right position.- The cassette must be placed on the transfer tank, minding that the black side of the cassette be placed closer to the black electrode (negative pole) and the red or white side of the cassette closer to the red electrode (positive pole).- Fill the tank with Transfer Buffer 1x. There must be enough buffer to cover up the entire cassette where the gel is placed. On the transfer tank there is a mark defining the specific height of buffer to be filled.* It is possible to transfer up to 4 gels simultaneously.- The lid of the transfer tank has a black and a red wire. Close the tank positioning the lid so that the black electrode connects to the black wire (negative pole) and the red or white electrode connects to the red wire (positive pole).- Connect the wires to the power supply, again placing the black wire to the black slot and the red wire to the red slot. Turn on the power supply and adjust it to 100V and 400mA for 1h. Alternatively, it might be transferred overnight on 10V (in this case, it is better to set up the transfer system into a fridge or a cold chamber, because the ice will melt before finishing the transfer). When turning on the power supply, it is necessary to observe whether the electric current is properly transmitted by checking the Amperage and the production of bubbles into the tank. - After the transfer, redraw carefully the cassette from the transfer tank. In the case of using a prestained molecular weight standard, this step must take into account whether the bands of the prestained standard have been transferred from the gel to the membrane. Transfer the membrane to a plastic or glass recipient with lid.- Stain membrane with 0.1% Amido Black Solution and leave the solution until the bands appear, homogenizing gently. This staining allows the protein bands (samples) to be seen in the membrane, i.e., whether the proteins have been transferred. With aid of tweezers, put the staining solution back to its recipient and mark the position of the molecular weight standard bands with a pencil (in the case of non prestained). Take a picture of the membrane, and then wash it with distilled water three times, 10 minutes each, homogenizing gently, to destain the membrane.Blocking: Since the membrane has affinity to any protein, this step blocks any free spot on the membrane preventing unspecific linkage of the antibody which will be used on the membrane.- Prepare circa 5 mL of 5% bovine serum albumin (BSA) in TBS-T, per membrane.* The blocking reagent must be enough to cover entirely the membrane.** Alternatively, BSA might be substituted by non-fat skimmed milk.- Incubate the membrane in the blocking solution for one hour, homogenizing gently.- Discard the blocking solution on the sink and wash the membrane by adding TBS-T solution enough to cover the membrane entirely, homogenizing gently for 10 minutes. Repeat the washing step twice.Antibody labeling:- Set up the primary antibody solution with TBS-T. The concentration of the primary antibody varies broadly among the different antibody stocks, thus it is necessary to check each corresponding datasheet regarding the recommended dilution. Typically, the dilution employed is 1:500 or 1:1000.* The antibody dilution is set up with 5% BSA on TBS-T.** The volume of the dilution must be enough to entirely cover the membrane. Since antibodies are expensive, set up minimum volume possible.- Incubate the membrane for at least one hour at room temperature on the primary antibody, homogenizing gently. The membrane might be kept overnight on this solution (in this case, maintain homogenizing into the fridge).- Discard the primary antibody solution and wash by adding TBS-T solution enough to cover entirely the membrane and leave homogenizing for 10 minutes. Repeat the wash twice. The primary antibody solution might be recovered and stored in a 50 mL flask in the fridge for further reuse.- Prepare the secondary antibody solution in TBS-T. In order to choose the secondary antibody, it is necessary to check in which animal the primary antibody was produced. The secondary antibody must recognize antibodies produced in the animal whence the primary antibody was made. For example, if the primary antibody was produced in mice, the secondary antibody must be anti-mouse. The secondary antibody concentration also depends on the type of antibody, but the dilutions are typically either 1:40,000 or 1:20,000.* It is necessary to set up enough volume to entirely cover the membrane. Since the antibodies are expensive, prepare the minimum volume.- Incubate the membrane in secondary antibody solution, homogenizing gently for one hour at room temperature.- Discard the secondary antibody solution, and wash the membrane adding TBS-T enough to entirely cover the membrane and homogenize for 10 minutes. Repeat the wash twice.- Discard the TBS-T on the sink and set up the development step.Development:- Bring to the dark chamber: Membrane in a recipient with TBS, tweezers, ECL Prime Western Blotting Detection Reagent, development cassette located under the electrophoresis bench, plastic film, scissors, 100μL micropipette, tips for 100μL micropipette, marker pen, 1 mL microtube, development film, development and fixer solutions.* At the dark chamber:- Close and lock the dark chamber door. Turn off the light and turn on the red light.- There are three plastic recipients on the bench of the dark chamber. Place on each the development solution, water, and fixing solution, respectively.- Prepare the substrate solution into a microtube, by mixing A and B solution 100μL each (read ECL kit instructions regarding the proper amount of each solution. Some kits have different recommendations).- Place the membrane using tweezers into the plastic film and then place it into the development cassette. Open the plastic film and, with the micropipette, spread over the whole membrane surface the substrate solution. Close the plastic film with the membrane and substrate inside. With a paper towel, spread the solution over the membrane eliminating bubbles.- Cut the development film with the same size as the membrane. Place the remaining film back into the box to avoid damaging it. Make a small cut in the corner to help with the identification of sample loading site. Place the film above the plastic and close the cassette. Keep the film exposed to the membrane during the desired time (depends on the antibody, but typically it is one to five minutes).- Open the cassette and with tweezers remove the film from the cassette placing it into the recipient with development solution. Move the recipient gently so that the solution interacts uniformly with the film. Incubate into the solution until the dark bands can be seen.- Remove the film from the development solution with tweezers and wash it into the recipient with water for two minutes.- Place the film into the recipient with fixer solution and move it gently until the film develops.- Wash the film in water for two minutes.- Still with the light off, return the development and fixer solutions to their bottles, be careful to not to switch them, and store all the remaining material before turning on the light.

Proper citation: Larissa Andrade, João Pedro Santos, María Cristina Vidal Pessolani 2018. Procedure for Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.pnrdmd6 Copy   

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Authors: Wen Aw
Group: Cage Studies

Proper citation: Wen Aw 2018. Acetyl-CoA Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.rv5d686 Copy   

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Authors: Lev Tsypin, Aaron Turkewitz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes how to deposit several cells of S. robusta in droplets on a petri dish, allowing the experimenter to track the growth of approximately single cell isolates with high throughput. This was used to determine drug sensitivity.We found that in, liquid culture, S. robusta is sensitive to 10 ug/mL puromycin and 250 ug/mL glyphosate. One caveat to keep in mind is that S. robusta appears more tolerant to antibiotics when grown on agar plates, being insensitive to up to 20 ug/mL of puromycin, for example. We recommend selection in liquid culture.

Proper citation: Lev Tsypin, Aaron Turkewitz 2019. Cell deposition and drug sensitivity assay. protocols.io https://dx.doi.org/10.17504/protocols.io.336gqre Copy   

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Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Invitrogen's HA tag monoclonal antibody in combination with Western Breeze Anti-Mouse Chemiluminescent Western Blot Immunodetection Kit leads to no background Western blots with Pt lysates. HA tag thus represents a tag of choice for (1) efficient and unambioguous protein expression analysis, (2) immunofluorescence experiments and (3) various IP and pull-down experiments in Pt and possibly other diatoms as well as other marine microeukaryotes.      

Proper citation: Jernej Turnsek 2017. HA tag enables highly efficient detection of heterologous proteins in Phaeodactylum tricornutum (Pt) exconjugants. protocols.io https://dx.doi.org/10.17504/protocols.io.j7ncrme Copy   

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Authors: Julia Mwesigwa
Summary:

Proper citation: Julia Mwesigwa 2017. DETECTION OF PLASMODIUM INFECTION BY NESTED POLYMERASE CHAIN REACTION ASSAY. protocols.io https://dx.doi.org/10.17504/protocols.io.i9gch3w Copy   

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Authors: avinash.kale
Group: UM-DAE Centre for Excellence in Basic Sciences

Proper citation: avinash.kale 2020. DNA extraction and Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bjdxki7n Copy   

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Authors: David Eccles
Summary: This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol

Proper citation: David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io https://dx.doi.org/10.17504/protocols.io.42dgya6 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the system that is used to capture Z stack images which are reassembled post-processing into 3D reconstructions.

Proper citation: Allen Institute for Brain Science 2020. Bright Field Image Capture with Zeiss AxioImager. protocols.io https://dx.doi.org/10.17504/protocols.io.bcsgiwbw Copy   

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Authors: Paul Whaley
Group: Systematic Reviews
Summary: A protocol template to help researchers follow the COSTER recommendations for conduct of systematic reviews. This instance covers the planning steps of a systematic review and will help with writing up the systematic review protocol.The intent is to convert COSTER from a checklist of things which need to be done into a sequence of actions which can be followed by a research team.When completing the protocol and either registering it or submitting it to a journal, please cite this instance of the protocol template and the parent manuscript, DOI 10.1016/j.envint.2020.105926.

Proper citation: Paul Whaley 2020. Generic Protocol for Environmental Health Systematic Reviews Based on COSTER Recommendations. protocols.io https://dx.doi.org/10.17504/protocols.io.biktkcwn Copy   

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Authors: Kerry Wiles
Group: Laboratory of Systems Pharmacology, NCIHTAN
Summary: The biosafety guidelines in this protocol are based on the Center for Disease Control's Biosafety in Microbiology and Biomedical Laboratories (BMBL), which has served as the cornerstone of biosafety practice in the United States since its initial release in 1984. The information in this protocol was extracted from the BMBL and truncated to provide CHTN VUMC staff members with the knowledge necessary to complete their training requirements. The protocol is used as both a reference and training SOP for the tissue procurement and the bodily fluids/blood processing protocols.

Proper citation: Kerry Wiles 2021. Tissue Procurement: Biosafety Guidelines. protocols.io https://dx.doi.org/10.17504/protocols.io.6y6hfze Copy   

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Authors: Alexander Culley, Grieg Steward
Group: VERVE Net, Sullivan Lab
Summary: Culley Protocol (from Culley & Steward (2007), New genera of RNA viruses in subtropical seawater, inferred from polymerase gene sequences, Applied & Environmental Microbiology 73(18):5937-5944).

Proper citation: Alexander Culley, Grieg Steward 2015. Can Crusher-Culley Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.c3jykm Copy   

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