DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), bovine liver phosphatidylinositol (PI), brain PS, and egg PA were purchased from Avanti Polar Lipids. PI 3-phosphate diC16 [PI(3)P], PI 4-phosphate diC16 [PI(4)P], and PI 5-phosphate diC16 [PI(5)P] were obtained from Echelon Biosciences. Folch fraction I and egg PC were purchased from Sigma-Aldrich. YM201636 PIKfyve inhibitor was purchased from Cayman Chemical. A cell fractionation kit was obtained from Cell Signaling Technology. Nontargeting control siRNA was obtained from Horizon Discovery, and human KLC1/2 siRNAs were from Santa Cruz Biotechnology. The following primary antibodies were used: horseradish peroxidase (HRP)–conjugated anti-His6 (71841, Novagen), anti-GFP for immunoblotting (3E1, Roche), anti-actin (AC-74, Sigma-Aldrich), anti–β-tubulin (AA2, Sigma-Aldrich), anti–histone H3 (Abcam), anti-giantin (PRB-114C, Covance), anti-HA (HA-7, Sigma-Aldrich), anti-KLC1 ([EPR12441(B)], Abcam), anti-KLC2 (Abcam), anti-KIF5B (Abcam), anti-LAMP1 (lysosome-associated membrane protein 1) (D2D11, Cell Signaling Technology), anti-Rab7 (D95F2, Cell Signaling Technology), and anti-Rab6 (D37C7, Cell Signaling Technology). Alexa 568– and Alexa 633–conjugated anti-mouse or anti-rabbit secondary antibodies were from Thermo Fisher Scientific.
Folch fraction 1
Manufactured by Merck Group
Folch fraction I is a laboratory product used for the extraction and purification of lipids from biological samples. It is a mixture of chloroform and methanol, commonly used in the Folch method for lipid extraction. The core function of Folch fraction I is to facilitate the separation and isolation of lipids from other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Anti lamp1, by Abcam (1 mentions)
Anti β tubulin aa2, by Merck Group (1 mentions)
Anti kif5b, by Abcam (1 mentions)
Anti rab7 d95f2, by Cell Signaling Technology (1 mentions)
Cell fractionation kit, by Cell Signaling Technology (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Non targeting control sirna, by Horizon Discovery (1 mentions)
Anti actin ac 74, by Merck Group (1 mentions)
Anti ha ha 7, by Merck Group (1 mentions)
Brain ps, by Avanti Polar Lipids (1 mentions)
Egg pa, by Avanti Polar Lipids (1 mentions)
Bovine liver phosphatidylinositol pi, by Avanti Polar Lipids (1 mentions)
Tla100.1 rotor, by Beckman Coulter (1 mentions)
Storm 820 phosphorimager, by GE Healthcare (1 mentions)
Gamma 32p atp, by PerkinElmer (1 mentions)
Pi 4 p, by Echelon Biosciences (1 mentions)
Odyssey system, by LI COR (1 mentions)
1011 electron microscope, by JEOL (1 mentions)
Whatman, by Cytiva (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
13 protocols using folch fraction 1
1
Endosomal Trafficking Lipid Composition
2
Lipid Tubulation Kinetics Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following synthetic lipids were used: POPG, POPE and POPC (Avanti Polar lipids). Additionally a bovine brain extract, Folch Fraction I (Sigma Aldrich) was used. For tubulation experiments large uni-lamellar vesicles (LUVs) or multi-lamellar vesicles (MLVs) were prepared. The dried lipid film, containing 2POPG:1POPE (w/w) was hydrated with buffer (20 mM Hepes pH 7.4) to achieve MLVs. LUVs were obtained by extrusion with a filter membrane (200 nm) (Avanti Polar lipids). The lipid condition was determined based on the screenings done by Isas et al.13 (link). All prepared lipids were either immediately used or stored at 4 °C for 2–3 days.
For the tubulation kinetics experiments various amphiphysin/BIN1 fragments (20 μM, 15 μM or 6 uM of N-BAR and N-BAR-deltaH0 or 6 μM of FL and deltaH0) were incubated with 180 μM of liposomes. The tubulation was measured by absorbance spectrometry at 400 nm and by negative-stain EM. For tubulation assays the absorbance was followed for 120 min. Additionally, aliquots of different proteins were taken at several time points up to 45 min and samples were assessed with negative-stain EM.
For the tubulation kinetics experiments various amphiphysin/BIN1 fragments (20 μM, 15 μM or 6 uM of N-BAR and N-BAR-deltaH0 or 6 μM of FL and deltaH0) were incubated with 180 μM of liposomes. The tubulation was measured by absorbance spectrometry at 400 nm and by negative-stain EM. For tubulation assays the absorbance was followed for 120 min. Additionally, aliquots of different proteins were taken at several time points up to 45 min and samples were assessed with negative-stain EM.
3
Liposome-Protein Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A liposome stock was prepared by resuspending bovine brain lipid extracts (Folch fraction I, Sigma B1502) with HEPES buffer (20 mM HEPES, 100 mM NaCl, 1 mM DTT, pH 7.0). The stock at 5 mg/mL was dissolved by ultrasonication on ice. Protein samples at 0.3 mg/mL (without tags) were incubated with liposomes at room temperature for 15 min and then centrifuged at 100,000 × g for 40 min at 4°C in a Beckman TLA100.1 rotor. After centrifugation, the supernatants were collected to determine the unbound proteins. The pellets were washed and resuspended with loading buffer to determine the bound proteins. Both the supernatant and pellet proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie blue staining.
4
Preparation and Characterization of Liposomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liposomes were prepared from a bovine brain extract, Folch Fraction I (Sigma Aldrich) by evaporating the chloroform of the lipid under a nitrogen-stream and further incubation under vacuum. Multi-lamellar vesicles (MLVs) were generated by hydrating the dried lipid with 20 mM Hepes, 150 mM NaCl, pH 7.5 to a concentration of 2 mg/ml. Large unilamellar vesicles were generated by extruding the MLV-suspension through filter (Avanti) with pore sizes of 200 nm, 100 nm, and 50 nm.
GUVs were generated using the gentle hydration method52 (link). Briefly, 100 µl of 10 mg/ml Folch Fraction I with 0.1% ATTO 655 labeled DOPE (ATTO-TEC) dissolved in 20:9:1 chloroform: methanol: H2O was dried on glass test tube under a nitrogen-stream and subsequent incubation under vacuum, then 1 ml of 10 mM Hepes, 240 mM sucrose, pH 7.5 was added gently to the glass tube without disturbing the lipid layers. The tube was incubated at 75 °C overnight. The bulky cloud floating in the middle of the solution, containing the GUVs, was transferred to 10 mM Hepes, 150 mM NaCl, pH 7.5 for fluorescence imaging.
GUVs were generated using the gentle hydration method52 (link). Briefly, 100 µl of 10 mg/ml Folch Fraction I with 0.1% ATTO 655 labeled DOPE (ATTO-TEC) dissolved in 20:9:1 chloroform: methanol: H2O was dried on glass test tube under a nitrogen-stream and subsequent incubation under vacuum, then 1 ml of 10 mM Hepes, 240 mM sucrose, pH 7.5 was added gently to the glass tube without disturbing the lipid layers. The tube was incubated at 75 °C overnight. The bulky cloud floating in the middle of the solution, containing the GUVs, was transferred to 10 mM Hepes, 150 mM NaCl, pH 7.5 for fluorescence imaging.
5
Characterization of zPIP5Kα Kinase Activity
6
Liposome Copelleting Assay with Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liposomes made from Folch fraction I (Sigma) were generated as described (Koch et al. 2011) . For liposome copelleting with purified protein, tag-free proteins were pre-spun at 200 000 g for 7 min at 28 • C before liposome binding. Pre-cleared, tagfree proteins (0.1 mg/ml) were then incubated with liposomes (1 mg/ml) in H-buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM DTT) for 15 min at RT and centrifuged at 200 000 g for 20 min at 28 • C. Supernatant (unbound) and pellet (bound) were boiled in SDS sample buffer and subjected to SDS-PAGE and Coomassie staining. Visualization was done using the LI-COR Odyssey system.
Liposome copelleting assays with membrane-depleted brain lysates were performed as described previously (Koch et al. 2011) .
Liposome copelleting assays with membrane-depleted brain lysates were performed as described previously (Koch et al. 2011) .
7
Lipid-binding Assay of NmPin Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipid-binding of NmPinwt, NmPinK7E/K34E and NmPinK7E was carried out as described previously with modifications [80 (link), 81 (link)]. Brain lipid extracts from bovine (Folch fraction I, Sigma) were resuspended in HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) to a concentration of 5 mg/mL under continuous stirring. The protein samples (15 μM) were incubated with varying liposome concentrations for 15 min at 37 °C and 350 rpm in a total volume of 40 μL and subsequently centrifuged (50 min, 100,000 × g, 4 °C). The supernatant was removed and the pellet resuspended in the equivalent volume HEPES buffer prior to analysis by SDS-PAGE.
8
Liposome Preparation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mixed brain liposomes—Folch fraction 1 (Sigma) were re-suspended in 20 mM HEPES (pH 7.4), 150 mM NaCl and 1 mM DTT to a final concentration of 1 mg ml−1. The mixture was then extruded using a 100 nm filter. Liposomes were stored at 4 °C and used within 4 days.
9
Liposome Formation for Lipid Experiments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
22 μl of a 25 mg/ml solution of Folch fraction-1 (Sigma) or a purpose made mixture of 65% PC, 15% PE, and either 20% PI(4)P, PI(3)P or PI(4,5)P2 (Avanti Polar Lipids dissolved in chloroform) was prepared, and dried under a nitrogen stream. Liposomes were formed by resuspension of the dried lipids in 200 μl F-buffer (0.2 mM CaCl2, 12 mM Tris/HCl, pH 8.0, 1 mM NaN3, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA) at 60°C for 30 mins with regular agitation.
10
Liposome Preparation from Folch Fraction 1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For preparation of liposomes 11 µl of a 25 mg/ml solution of Folch fraction 1
(Sigma) was dried under nitrogen, then resuspended in 200 µl of buffer B (20
mM HEPES pH 7.2, 100 mM KCl, 2 mM MgCl2, 1 mM DTT) at 60°C for 30
min with gentle agitation. Liposomes were extruded 11 times through
polycarbonate filters with 1.0 µm pores.
(Sigma) was dried under nitrogen, then resuspended in 200 µl of buffer B (20
mM HEPES pH 7.2, 100 mM KCl, 2 mM MgCl2, 1 mM DTT) at 60°C for 30
min with gentle agitation. Liposomes were extruded 11 times through
polycarbonate filters with 1.0 µm pores.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!