Drosophila S2 cell samples were boiled for 10′ in loading buffer separated on 6% (Spt6) or 10% (Co-IPs) SDS-PAGE gels and processed for western blotting using mouse α GFP (1:2000, Clone 71; D. van Essen), monoclonal mouse α Spt6 (25C6, Helmholtz Zentrum München, IgG2b 1:500) was raised against Spt6 amino acid residues 215–230 DYDDFSKYEEDDYEDD, rabbit α CENP-A (1:5000, ab10887, Abcam), mAB α Flag (Clone M2; 1:10000, F1804, Sigma-Aldrich), rabbit α H3 (1:10000, ab1791, Abcam) and mouse α tubulin AA4.3 (1:1000; DSHB). Secondary polyclonal Goat antibodies (Sigma) coupled to horseradish peroxidase Rabbit IgG HRP Linked Whole Ab, (NA934) and Mouse IgG HRP Linked Whole Ab (GE Healthcare, NA931) were used at 1:10000. HeLa cells were boiled in 2× Laemmli sample buffer and whole cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes using a Trans Blot System (BioRad). Antibodies used were SPT6 (Abcam, ab32820 rabbit polyclonal; 1:500), CENP-C (Gift from D. Cleveland UCSD, made in house from Covance clone #3024, rabbit polyclonal; 1:10000) and α-tubulin (Sigma, T9026 mouse monoclonal; 1:1000). Secondary antibodies used were Donkey anti-Rabbit 800 (Li-Cor, 926–32211) and Donkey anti-mouse 680 (Rockland, 610-744-124; both at 1:10000). Blots were visualized using Image Lab 6.1 (BIO RAD).
Mouse igg hrp linked whole ab
Manufactured by GE Healthcare
The Mouse IgG HRP Linked Whole Ab is a laboratory equipment product. It is a secondary antibody that is conjugated with horseradish peroxidase (HRP). This product can be used for various immunoassay applications that require the detection of mouse immunoglobulin G (IgG).
Automatically generated - may contain errors
Lab products found in correlation
Rabbit igg hrp linked whole ab, by GE Healthcare (3 mentions)
T9026, by Merck Group (1 mentions)
F1804, by Merck Group (1 mentions)
Trans blot system, by Bio-Rad (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab10887, by Abcam (1 mentions)
Ab32820, by Abcam (1 mentions)
Donkey anti rabbit 800, by LI COR (1 mentions)
Donkey anti mouse 680, by Rockland Immunochemicals (1 mentions)
Hif 1α, by BD (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Rpn2106, by GE Healthcare (1 mentions)
Na943, by GE Healthcare (1 mentions)
Ab8226, by Abcam (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Lovastatin, by Merck Group (1 mentions)
Temozolomide, by Merck & Co (1 mentions)
Gtu 88, by Merck Group (1 mentions)
6 protocols using mouse igg hrp linked whole ab
1
Western Blot Analysis of Drosophila and HeLa Cells
2
Analysis of E2F and HIF-1α Proteins
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Whole-cell lysates were prepared using a lysis buffer containing 150 mM NaCl, 1.0% NP40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris (pH 8.0), supplemented with Protease Inhibitor Cocktail (Roche). Protein lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Membranes were probed with the following antibodies: E2F7 (Santa Cruz, sc-66870), E2F8 (Abnova, H00079733-M01; Abcam AB109596), HIF1α (BD Biosciences, 610959), HDAC1 (sc-7872), E2F1 (sc-193), Mouse IgG HRP-linked whole Ab (GE Healthcare, NA931), Rabbit IgG HRP-linked whole Ab (GE Healthcare, NA934). As secondary antibodies, anti-rabbit-HRP (Amersham Biosciences, NA934; 1:5000) and anti-mouse-HRP (Amersham Biosciences, NA931; 1:5000) were used. All antibodies were diluted in 4% non-fat milk in TBST. Immuno-probed blots were subjected to standard ECL reagents as described by the manufacturer (GE Healthcare, RPN2106).
3
Western Blot Analysis of KLF4 and β-Actin
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Western blot analysis was performed as described previously [24 (link)]. Primary antibodies used were rabbit polyclonal KLF4 antibody (1:2,000; GTX101508; GeneTex) and mouse monoclonal β-actin antibody (1:2,000; ab8226; Abcam). Secondary antibodies used were rabbit IgG HRP Linked Whole Ab (1:10,000; NA943; GE Healthcare) and mouse IgG HRP Linked Whole Ab (1:10,000; NA941; GE Healthcare).
4
Temozolomide and Lovastatin Cytotoxicity Assay
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Temozolomide (Schering-Plough, NJ, USA) was dissolved in DMSO to make a stock of 10 mM. Lovastatin was obtained from Sigma (St. Louis, MO, USA) and dissolved in DMSO at a concentration of 10 mM. Thiazolyl blue tetrazolium bromide (MTT), DMSO, and bafilomycin-A1 were obtained from Sigma. The primary antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA) and Santa Cruz Biotechnology (SC, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) and diluted in 5% bovine serum albumin (BSA) according to the manufacturer's instructions: SQSTM1/p62 (CST #5114, 1 : 1000), LC3A/B (CST #12741, 1 : 1000), cleaved caspase-3 (CST #9661, 1 : 1000), cleaved PARP (CST #5625, 1 : 1000), Bcl-2 (CST #4223, 1 : 1000), Bim (CST #2933, 1 : 1000), Akt (CST #4691, 1 : 1000), p-AKT (CST #4060, 1 : 1000), mTOR (CST #2983, 1 : 1000), GAPDH (CST #5174, 1 : 1000). LAMP1 (SC-20011, 1 : 1000), LAMP2 (SC-18822, 1 : 1000), and dynein (SC-514579, 1 : 1000). The secondary antibodies included anti-rabbit IgG, HRP-linked antibody (CST #7074, 1 : 5000), and mouse IgG HRP linked whole Ab (GE Healthcare, NA931, 1 : 5000).
5
Western Blot Analysis of Protein Expression
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Cells were lysed in lysis buffer (50 mM Tris-HCl -pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton-X, 1mM DTT, 1 mM PMSF, and supplemented with complete protease inhibitor mix (Roche),) for 30 min on ice, followed by centrifugation at maximum speed for 10 min at 4°C. An equal volume of 4x Laemmli buffer was added to the supernatants and the samples were boiled for 5 min. Approximately 1x10 5 cells were separated on an SDS-PAGE gel, followed by blotting to nitrocellulose membranes. The membranes were blocked in 5% BSA in TBST, incubated with primary antibodies, followed by an incubation with horseradish peroxidase (HRP) secondary antibodies (Mouse IgG HRP Linked Whole Ab-GE Healthcare, #NA931, 1:5000; Rabbit IgG HRP Linked Whole Ab-GE Healthcare, #NA934, 1:5000) in 3% milk in TBST. Primary antibodies: M2 Flag (Sigma Aldrich, 1:10,000 dilution), anti-c-Myc antibody clone 9E10 (Millipore, 1:1000 dilution), anti-HA.11 clone 16B12 (Biolegend, previously Covance Catalog# MMS-101P, 1:1000 dilution) and gamma-tubulin clone GTU-88 (Sigma Aldrich, 1:5000 dilution). Streptavidin-HRP (Invitrogen, #1953050) was used for detection of biotinylated proteins.
6
Western Blot Analysis of Zebrafish Embryos
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Dechorionated zebrafish embryos were de-yolked in Ginzburg buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3) and lysed in Laemli buffer (20 embryos in 80 ul). Either 10 or 20 ul of sample were separated by 12 % SDS-PAGE and transferred onto PVDF membrane (Millipore).
After blocking with 5 % Milk powder (Roth) in TBST, membranes were incubated with antiphospho-p44/42 MAPK (Thr202/Tyr204) (1:1000; #8544; Cell Signaling), anti-p44/42 MAPK antibody (1:2000; #9102, Cell Signaling) and anti-αActin antibody (1:5000; A-5060, Sigma), Phospho-Akt (Ser473) (1:2000; #4060; Cell Signaling), Akt (1:1000; #9272; Cell Signaling).
Primary antibodies were detected using mouse IgG HRP linked whole Ab (1:4000; NXA931; GE healthcare).
After blocking with 5 % Milk powder (Roth) in TBST, membranes were incubated with antiphospho-p44/42 MAPK (Thr202/Tyr204) (1:1000; #8544; Cell Signaling), anti-p44/42 MAPK antibody (1:2000; #9102, Cell Signaling) and anti-αActin antibody (1:5000; A-5060, Sigma), Phospho-Akt (Ser473) (1:2000; #4060; Cell Signaling), Akt (1:1000; #9272; Cell Signaling).
Primary antibodies were detected using mouse IgG HRP linked whole Ab (1:4000; NXA931; GE healthcare).
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