Ub-V5 conjugated U2AF2-Flag was purified from HEK293T cells with anti-Flag M2 Affinity Gel (Sigma-Aldrich, Saint Louis, Missouri, USA). The protein complexes containing HA tagged wild-type OTUB2 or the C51S mutant type of OTUB2 were purified from HEK293T cells with anti-HA Affinity Gel (Sigma-Alrich). UB-HA-U2AF2 and the OTUB2 protein complexes were incubated for 1 h at 37 °C in the reaction buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 100 mM NaCl, 1 mM ATP). After the reaction, the U2AF2-Flag protein was purified and immunoblotted with antibodies against V5.
Anti ha affinity gel
Manufactured by Merck Group
Sourced in United States
Anti-HA affinity gel is a laboratory product designed for the purification and detection of proteins that are tagged with the hemagglutinin (HA) epitope. It is used to isolate and concentrate HA-tagged proteins from complex samples, such as cell lysates or culture supernatants.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 affinity gel, by Merck Group (6 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Ha peptide, by Merck Group (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Anti flag affinity gel, by Merck Group (3 mentions)
Anti flag, by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
35s methionine, by Promega (2 mentions)
Tnt quick coupled transcription translation system, by Promega (2 mentions)
Anti myc affinity gel, by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Ez view red protein a g, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Mg132 s2619, by Selleck Chemicals (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti phosphoserine, by Thermo Fisher Scientific (1 mentions)
Anti akt sc 8312, by Santa Cruz Biotechnology (1 mentions)
Anti myc 16 286 1 ap, by Proteintech (1 mentions)
Anti ha 51064 2 ap, by Proteintech (1 mentions)
35 protocols using anti ha affinity gel
1
OTUB2 Deubiquitination of U2AF2
2
Co-immunoprecipitation of HA-H11 and FLAG-Vpx
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HEK293 cells were co-transfected with HA-H11 and FLAG-Vpx, and treated with 20 μM of MG132 for 4 h before harvest. Harvested cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 mM DTT) containing complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA) and PhosSTOP phosphatase inhibitor cocktail (Roche Molecular Biochemicals). Lysates were cleared by centrifugation at 12,000 × g for 15 min, followed by pull-down with anti-FLAG M2 affinity Gel (Sigma) or anti-HA affinity Gel (Sigma). Samples were separated by SDS-PAGE and analyzed by Western blotting.
3
Antibody Immunoblotting Protocol
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Commercial antibodies used for western blotting analysis include: anti-Phospho-Akt (Ser473) (#3787, 1:1000 dilution), anti-Phospho-Akt Substrate (R××pS/pT) (#9614, 1:1000 dilution for WB and 1:250 dilution for IP), anti-USP14 (rabbit, #11931, 1:1000 dilution), anti-FLAG (#2368, 1:1000 dilution) were from Cell Signaling Technology. Anti-phosphoserine (#61-8100, 1:1000 dilution for WB and 1:250 dilution for IP) was from Invitrogen. Anti-Akt (sc-8312, 1:1000 dilution) and anti-USP14 (mouse, sc-393872, 1:1000 dilution) were from Santa Cruz Biotechnology. Anti-Myc (16286-1-AP, 1:1000 dilution), anti-HA (51064-2-AP, 1:1000 dilution), and anti-GAPDH (60004-1-Ig, 1:10000 dilution) were from Proteintech. Anti-Tubulin (PM054, 1: 10000 dilution) was from MBL. Recombinant active Akt protein (#14-276) was from Millipore. Mouse monoclonal anti-FLAG (F1804, 1:500 for IP), anti-Myc (A7470), and anti-HA Affinity Gel (E6779) were from Sigma-Aldrich. IU1 (S7134), MK2206 (S1078), AZD5363 (S8019), GDC0941(S1065), Wortmannin (S2758), U0126 (S1102), and MG132 (S2619) were from Selleckchem. Phospho-USP14 S432 antibodies were generated by Proteintech. Briefly, synthetic peptides corresponding to phosphorylated S432 epitope (N'-Cys-THQGRSSs(phospho)SGHYVSW) of USP14 were used to immunize rabbits and the antisera were affinity purified.
4
Immunoprecipitation of Tagged Proteins
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293 T cells were lysed in ice-cold NP40 lysis buffer supplemented with a protease inhibitor cocktail, and centrifuged (12,000 × g) at 4 °C for 10 min. The cell lysates were used for immunoprecipitation with Pierce Crosslink Immunoprecipitation Kit (26,147, Thermo Scientific), anti-Flag M2 affinity gel (A2220, Sigma–Aldrich), or anti-HA affinity gel (E6779, Sigma–Aldrich). The obtained beads were washed five times with lysis buffer, and the immunoprecipitates were obtained by boiling in 2 × loading buffer at 95 °C for 5 min, or eluted by 100 μg/mL HA peptide (I2149; Sigma–Aldrich). Finally, the immunoprecipitates and whole cell lysates were subjected to immunoblotting using the indicated primary antibodies and corresponding secondary antibodies. The details of antibodies are listed in Table S1 .
5
Co-IP Assay for Drosophila S2 Cells
6
mTOR Regulation by Chaperone Proteins
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MTT, MG132, and chloroquine were purchased from Sigma-Aldrich (St Louis, MO, USA). Rabbit antibodies to mTOR, p-mTOR (S2481), Akt, p-Akt (S473), CDK4 (D9G3E), ubiquitin, Cdc37 (D11A3), and Hsp90, were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Flag (M2), anti-HA, anti-Flag M2 affinity agarose gel, and anti-HA affinity gel were purchased from Sigma-Aldrich. Celastrol was purchased from Aladdin (Shanghai, China). pcDNA3-Flag-mTOR was a gift from Jie Chen (Addgene plasmid 26603). pcDNA3-HA-Hsp90α and pcDNA3-HA-Cdc37 were gifts from Dr Len Neckers (National Institutes of Health, USA). All inserts in the constructs were verified by DNA sequencing.
7
Analysis of Fhit-Gα16/z Interactions
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The human cDNAs of various Gα subunits were obtained from Guthrie Research Institute (Sayre, PA). Wild-type Fhit in pCMV-SPORT6 was purchased from Invitrogen (Carlsbad, CA). Gα16/z chimeras were constructed by overlapping PCR which swapped the corresponding regions of Gα16 with Gαz as described previously [15 (link)]. Cell culture and Lipofectamine PLUS reagents, and anti-Fhit antibody were purchased from Invitrogen (Carlsbad, CA). Anti-Gα16 was obtained from Gramsch Laboratories (Schwabhausen, Germany). Anti-Gαq/11 antibody was purchased from Calbiochem (San Diego, CA). Anti-α-tubulin, anti-HA, and anti-Flag antibodies as well as anti-HA affinity gel were from Sigma-Aldrich (St. Louis, MO). Antisera against PLCβ1/2/3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other antibodies were purchased from Cell Signaling Technology (Danvers, MA). Protein G-agarose was from Thermo Fisher Scientific (Rockford, IL). ECL kit was from Amersham Biosciences (Piscataway, NJ).
8
In Vitro Expression of PSA and PSMA Antigens
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Example 8
In vitro translation performed to confirm the expression of the PSA and PSMA antigens. The TNT® Quick Coupled Transcription/Translation System and 35S-methionine (Promega) were used. The pVAX vector alone (negative control) or pVAX backbone with the PSA or PSMA antigen inserts and 35S-methionine was added to the reaction mixture according to the manufacturer's instructions. The reaction was carried out at 30° C. for 2 bents. Labeled proteins were immunoprecipitated with anti-HA Affinity Gel (Sigma, St. Louis, Mo.) by rotation overnight in radioimmunoprecipitation assay (RIPA) buffer at 4° C. The immunoprecipitated proteins were electrophoresed on a SDS-PAGE gel that was subsequently fixed and dried. Expression of the 35S-labeled proteins was detected by autoradiography. The results are shown in
9
Isolation and Purification of Mitochondrial Proteins
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Isolated mitochondria (1 mg) were solubilized in digitonin-containing buffer (1% [wt/vol] digitonin, 10% [wt/vol] glycerol, 20 mM Tris-HCl, pH 7.4, and 300 mM NaCl) for 20 min on ice. After clarification of the solubilized material, the extracts were subjected to anti-HA Affinity Gel (Sigma-Aldrich, St. Louis, MO) for 1.5 h at 4°C, followed by washing with buffer A (20 mM Tris-HCl, pH 7.4, and 300 mM NaCl). The elution of bound material was performed by incubation in Laemmli buffer with 50 mM DTT. The eluted extracts were analyzed by reducing SDS–PAGE, followed by Western blot.
10
In vitro Validation of PSA and PSMA Antigens
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Example 8
In vitro translation performed to confirm the expression of the PSA and PSMA antigens. The TNT® Quick Coupled Transcription/Translation System and 35S-methionine (Promega) were used. The pVAX vector alone (negative control) or pVAX backbone with the PSA or PSMA antigen inserts and 35S-methionine was added to the reaction mixture according to the manufacturer's instructions. The reaction: was carried out at 30° C. for 2 hours. Labeled proteins were immunoprecipitated with anti-HA Affinity Gel (Sigma, St. Louis, Mo.) by rotation overnight in radioimmunoprecipitation assay (RIPA) buffer at 4° C. The immunoprecipitated proteins were electrophoresed on a SDS-PAGE gel that was subsequently fixed and dried. Expression of the 35S-labeled proteins was detected by autoradiography. The results are shown in
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