Stored blood was washed 5–7 times with PBS at 30,000 ×g for 25 min to remove hemoglobin. The pellets were resuspended in lysis buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 1% Nonidet P-40) and sonicated for 2 min in 30-s intervals. The samples were boiled in SDS-PAGE buffer for 5 min. Isolation of proteins from ISE6 cells infected with the St. Maries strain and uninfected ISE6 cells was done as previously described (Ramabu et al., 2010 (link)). Protein samples were electrophoresed on precast 12.5% Criterion Tris-HCL Gels (Bio-Rad) at 100 V for 3 h. After transfer to nitrocellulose and overnight blocking in I-Block reagent (Applied Biosystems) with 0.5% Tween 20, proteins were detected using the previously described monoclonal antibodies ANAF16C1 at 0.02 μg/ml for Msp5 and ANAO24D1 at 3 μg/ml for Aaap proteins (Eriks et al., 1994 (link)). Msp5, a constitutively expressed protein, was detected as a control to demonstrate equal loading in each lane. Antigen binding was detected with a 1:7000 dilution of alkaline phosphatase-conjugated goat anti-mouse secondary antibody from Western-Star chemiluminescent immunoblot detection systems (Applied Biosystems) according to manufacturer's specifications.
Criterion tris hcl gel
Manufactured by Bio-Rad
Sourced in United States
The Criterion Tris-HCl gels are pre-cast polyacrylamide gels designed for protein electrophoresis. They are available in different percentages of acrylamide to accommodate a range of protein molecular weights. The gels provide a consistent and reliable platform for separating and analyzing protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Chemidoc mp system, by Bio-Rad (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Criterion cell, by Bio-Rad (3 mentions)
Express plus membrane, by Merck Group (3 mentions)
Bio safe coomassie stain, by Bio-Rad (3 mentions)
Anti rabbit igg hrp conjugate, by Cell Signaling Technology (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Western lightning plus ecl chemiluminescence substrate, by PerkinElmer (3 mentions)
I block reagent, by Thermo Fisher Scientific (2 mentions)
Western star chemiluminescent immunoblot detection systems, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Superblock buffer, by Thermo Fisher Scientific (2 mentions)
Anti myc tag 9b11, by Cell Signaling Technology (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sodium dihydrogen phosphate, by Merck Group (2 mentions)
Glycin, by AppliChem (2 mentions)
Dribulose 1 5 diphosphate carboxylase, by Merck Group (2 mentions)
Coomassie brilliant blue g 250, by Serva Electrophoresis (2 mentions)
50 protocols using criterion tris hcl gel
1
Protein Isolation and Detection from Tick Cells
2
Protein Extraction and Detection from Tick Cells
3
Western Blot Analysis of HO-1 and GCLC
4
Protein Cross-Linking and Detection
5
Western Blot Analysis of Protein Lysates
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An aliquot of the total lysates was used for western blotting (WB). 0.1% SDS was added to the lysates, followed by sonication (3 cycles, 10 s each). Samples were run on 4–20% Criterion Tris-HCl gels (Bio-Rad Laboratories) and electrophoretically transferred to a nitrocellulose membrane (Thermo Fisher Scientific). Residual protein-binding sites were blocked by incubation with 5% non-fat milk in 1XTBST (1X TBS plus 0.1% Tween 20) 1 h at RT, followed by an overnight (O/N) incubation at 4 °C with primary antibodies diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBST. Mouse anti-tubulin (Thermo Fisher Scientific) were diluted 1:5000; anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was diluted 1:1000. Appropriate isotypes secondary antibodies HRP-conjugated were diluted 1:2000 or 1:10000 in 5% non-fat milk 1XTBST, and added for 1 h at RT. Every step was followed by 3 or 4 washes in 1X TBST. Detection was performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific) and exposed to x-ray films.
6
Quantification of PGRN Protein Levels
7
Immunoblotting of Cellular Proteins
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An aliquot of the total lysates was used for immunoblotting. 0.1% SDS was added to the lysates, followed by sonication (3 cycles, 10 s each). Samples were run on 4–20% Criterion Tris-HCl gels (Bio-Rad Laboratories) and electrophoretically transferred to a nitrocellulose membrane (Thermo Fisher Scientific). Residual protein-binding sites were blocked by incubation with 5% non-fat milk in 1XTBST (1X TBS plus 0.1% Tween 20) 1 h at RT, followed by an O/N incubation at 4 °C with primary antibodies diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBST. Mouse anti-N-Cadherin A60 (BD, Franklin Lakes, NJ), mouse anti-actin Ab5 (BD) were diluted 1:5000; anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was diluted 1:1000. Appropriate isotypes secondary antibodies HRP-conjugated were diluted 1:2000 in 5% non-fat milk 1XTBST, and added for 1 h at RT. Every step was followed by 3 or 4 washes in 1X TBST. Detection was performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific) and exposed to x-ray films.
8
Western Blot Analysis of Protein Interactions
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Samples from pull-down assays were analysed by SDS/PAGE on 4–15% or 15% Criterion Tris/HCl gels (Bio-Rad Laboratories) and blotted on to PVDF membranes (GE Healthcare). The PVDF membranes and peptide arrays were blocked in 5% (w/v) dried non-fat skimmed milk powder or 1% (w/v) casein in TBS-T for 60 min at room temperature and incubated overnight with primary antibody at 4°C. After incubation with primary antibody, the membranes were washed five times for 5 min in TBS-T and incubated further with an HRP (horseradish peroxidase)-conjugated secondary antibody. The membranes were developed using ECL Plus (GE Healthcare). The chemiluminescence signals were detected using a LAS 1000 or LAS 4000 instrument (Fujifilm).
9
Immunoprecipitation and Western Blot Analysis
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FLAG-IPs were performed as in Cugusi et al. (23 (link)). For immunoprecipitations with endogenous proteins, 2 mg of S2 cell extracts were incubated with 2 to 10 μl of a specific antibody or generic anti-mouse IgG (Jackson IR), overnight at 4 °C. Protein G Agarose Beads (Millipore, Billerica, MA) pre-equilibrated in lysis buffer were then added for two hours at 4 °C. After washing four times in PBS buffer containing 1% Tween-20, the beads were resuspended in loading buffer. Samples were loaded in Criterion Tris-HCl gels (Bio-Rad, Hercules, GA) and transferred to a polyvinylidene difluoride membrane by using 10% methanol-Tris-glycine transfer buffer, following Bio-Rad's Criterion protocol. The blots were probed using antibodies against, MLE (1:3000), Hrb87F/P11 (1:200), PEP/X4 (1:200), Mi-2 (1:2000), MEP-1 (1:2000), p66 (1:1000), MSL1 (1:3000), Topo II (Santa Cruz Biotechnology T22C5 1:200), and with appropriate secondary antibodies conjugated to HRP. Detection was recorded on x-ray films by chemiluminescence using ECL Prime Western blotting detection reagent (Amersham Biosciences # RPN2232). MEP-1 and Mi-2 antibodies were a gift from C.P. Verrijzer, p66 antibody was a gift from R. Nusse, Hrb87F and PEP antibodies were a gift from H. Saumweber.
10
Aspergillus oryzae Protoplast Transformation
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Example 5
Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422, which were transformed with approximately 2 μg of pSMai207. The transformation yielded about 30 transformants. Ten transformants were isolated to individual Minimal Medium plates.
Confluent Minimal Medium plates of each of the transformants were washed with 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of M410 medium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm. After 5 days incubation, 5 μl of supernatant from each culture were analyzed on CRITERION® Tris-HCl gels (Bio-Rad Laboratories, Hercules, Calif., USA) with a CRITERION® Cell (Bio-Rad Laboratories, Hercules, Calif., USA), according to the manufacturer's instructions. The resulting gels were stained with BIO-SAFE™ Coomassie Stain (Bio-Rad Laboratories, Hercules, Calif., USA). SDS-PAGE profiles of the cultures showed that the majority of the transformants had an expected 24 KDa band size. A confluent plate of transformant 3 was washed with 10 ml of 0.01% TWEEN® 80 and inoculated into a 2 liter Fernbach containing 500 ml of M410 medium to generate broth for characterization of the enzyme. The culture was harvested on day 5 and filtered using a 0.22 μm EXPRESS™ PLUS Membrane (Millipore, Billerica, Mass., USA).
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