We immunoprecipitated αB-crystallin from 100 μg of WS protein (human 9-year-old lens) using 100 μg of the αB-crystallin monoclonal antibody and treated with Protein A-Agarose (40 μL, Santa Cruz Biotechnology), centrifuged at 1000g for 5 min and used the supernatant in Western blotting for αB-crystallin or SuccK. The sample was processed similarly but without the antibody served as a control.
Protein a agarose
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Protein A agarose is a solid-phase affinity resin used for the purification of antibodies. It consists of the Protein A ligand covalently coupled to an agarose bead matrix. Protein A is a bacterial protein that binds specifically to the Fc region of immunoglobulins, allowing antibodies to be captured and purified from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Cell lysis buffer, by Beyotime (3 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (3 mentions)
Bovine serum albumin (bsa), by Roche (3 mentions)
Bl003a, by Biosharp (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Protein a agarose, by Thermo Fisher Scientific (3 mentions)
Phenylmethanesulfonyl fluoride, by Dingguo (2 mentions)
Anti ha, by Abcam (2 mentions)
Anti pan phospho sr, by Santa Cruz Biotechnology (2 mentions)
Anti srpk1, by Santa Cruz Biotechnology (2 mentions)
Anti flag tag, by Abcam (2 mentions)
Anti srsf1, by Abcam (2 mentions)
Percoll density gradient media, by GE Healthcare (2 mentions)
Rpmi 1640 medium, by Sartorius (2 mentions)
Lactate assay kit, by Nanjing Jiancheng (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Agarose, by Santa Cruz Biotechnology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
77 protocols using protein a agarose
1
Immunoprecipitation of αB-Crystallin from Lens
2
Immunoprecipitation and Western Blotting for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
COS-7 cells overexpressing ROP18I and/or NMI (IL20RB, P2RX1, IL21, or vimentin) were prepared as mentioned previously in the FRET assay. Cell extracts were prepared by lysing the cells in cell lysis buffer (#P0013, Beyotime, Shanghai, China) with 1 mM phenylmethanesulfonyl fluoride (#WB-0181, Beijing Dingguo Changsheng Biotechnology, Beijing, China). Cell lysates were incubated with the primary antibody (anti-NMI rabbit monoclonal antibody anti-HA rabbit monoclonal antibody, anti-P2RX1 goat polyclonal antibody, or anti-FLAG mouse monoclonal antibody) with gentle rotation for 1 h at 4°C. Protein A-Agarose (#sc-2001, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was then added to the immunoprecipitation reaction with incubation overnight at 4°C. The immunoprecipitates were washed four times with phosphate-buffered saline and then eluted by boiling with SDS-PAGE loading buffer (#9173, TAKARA, Kusatsu, Japan). The eluates were analyzed by western blotting with the indicated antibodies, as described previously (28 (link)). For the UBC experiment, cells were treated with 10 μM proteasome inhibitor MG132 (#S1748, Beyotime, Shanghai, China) for 12 h before harvesting.
3
USP39-SRPK1/SRSF1 Interaction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells with stable overexpression or knockdown of USP39 were collected by RIPA buffer. Immunoprecipitation was conducted with anti-HA (Abcam) or anti-SRPK1 (Santa Cruz) or anti-Pan-phospho-SR (Santa Cruz). By incubation with protein A agarose (Santa Cruz), the antibodies were removed. Proteins were prepared and separated by 10% SDS-PAGE. The interaction between USP39 and SRPK1/SRSF1 was analyzed by Western blot using anti-Flag tag (Abcam) or anti-SRSF1 (Abcam).
4
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates prepared in the buffer containing 150mM NaCl, 50mM Tris-HCl (pH 8.0), and 0.5% NP40 were mixed with protein A agarose (Santa Cruz) and the indicated antibodies at 4ºC overnight with rotation. The precipitated proteins were then eluted by boiling beads in sodium dodecyl sulfate (SDS)-loading buffer [4% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.2% bromophenol blue, 100mM Tris-HCl (pH 6.8)] and analyzed using Western blot.
5
Purification and Immunization of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain purified recombinant proteins, BL21 (DE3) E. coli transformed with pGEX-UBC9, pGEX-VP55, or pGEX-VP55M were cultivated in Luria-Bertani medium containing 100 μg ampicillin/mL at 37°C until the optical density reached 0.4-0.6 at 600 nm. Expression of the GST fusion proteins was induced using 0.1-0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 4-6 h. The pellet was collected by centrifugation at 5000× g for 5 min and resuspended in PBS (140 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4). The expressed protein was purified as previously described [42 (link)] or using the Glutathione Sepharose™ 4B kit instructions (GE Healthcare). Purified GST-VP55 was used to prepare polyclonal antiserum. Three New Zealand rabbits were intracutaneously immunized with 250 μg of GST-VP55 in Freud's adjuvant (Sigma, USA) at 2 week intervals for three times. Immunoglobulin (IgG) specific for VP55 was purified using Protein A Agarose (Santa Cruz).
6
EPHA2 Ubiquitination Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation (IP) was performed using anti-EPHA2 (#6997; CST) or anti-FLAG (#F1804; Sigma) antibody. Total protein (100 µg) was precleared using Protein A agarose (sc-2003, Santa Cruz Biotechnology) for 2 h at room temperature with slow rotation. Next, 1 μg of anti-EPHA2 or anti-FLAG antibody was used to probe the precleared protein by mixing overnight at 4 °C; this step was followed by centrifugation at 1,000 ×g for 1 min, and the supernatant was discarded. The pulled beads were washed three times with cold lysis buffer, and the proteins were eluted by boiling in 2X SDS loading buffer. Western blots for checking the expression of EPHA2, Ub, Ub(K48), and Ub(K63) were performed, as described above.
7
Isolation and Characterization of Murine Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli LPS (0111:B4), FITC-conjugated LPS from E. coli 0111:B4 (FITC-LPS) and paraformaldehyde (PFA) was from Sigma-Aldrich (St. Louis, MO). Collagenase Type 4 was from Worthington biochemical Corporation (Lakewood, NJ). Percoll density gradient media was from GE Healthcare (Uppsala, Sweden). Normal goat serum was from Jackson ImmunoResearch (West Grove, PA). Trizol reagent was from Life Technology (Carlsbad, CA). SYBR Green Master and bovine serum albumin (BSA) was from Roche (Basel, Switzerland). DMEM medium and fetal bovine serum (FBS) was from Gibco (Shelton, CT). High-sig ECL western blotting substrate was from Tanon (Shanghai, China). Protein A-Agarose was from Santa Cruz Biotechnology (Dallas, TX). Lactic acid dehydrogenase (LDH) assay kit was from Jiancheng Biotech (Nanjing, China).
8
Affinity Purification of Influenza Viral Ribonucleoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NP, PB2, FLAG-tagged PB1, FLAG-tagged PA, and segment 6 vRNA (NA) were expressed in transfected HEK 293T cells for 48 hr, following prior approaches [54 (link)]. Cells were lysed in co-IP buffer in the presence of protease inhibitors. Lysates were clarified by centrifugation, pre-cleared protein A agarose (Santa Cruz Biotech sc-2001) for 1 hr, and transferred to a new tube where BSA was added to a final concentration of 5 mg/mL. NP was immunoprecipitated overnight with 3 μg anti-NP antibody. Immunocomplexes were captured using protein A agarose (Sigma P2545) for 1 hr, washed twice with co-IP buffer containing 5 mg/mL BSA and 500 mM NaCl, and twice with co-IP buffer. Bound proteins were eluted by boiling in Laemmli buffer. Samples were then assayed via western blot analysis for presence of NP, PB1, and PA.
9
Optimized Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Modified McCoy's 5A medium and MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reagent were from Sigma-Aldrich (St. Louis, MO). Trizol reagent and Lipofectamine 3000 were from Life Technology (Carlsbad, CA). Bovine serum albumin (BSA) was from Roche (Basel, Switzerland). Normal Goat Serum was from Jackson ImmunoResearch (West Grove, PA). RPMI 1640 medium and fetal bovine serum (FBS) were from Biological Industries (Kibbutz Beit Haemek, ISRAEL). High-sig ECL western blotting substrate was from Tanon (Shanghai, China). Protein A-Agarose was from Santa Cruz Biotechnology (Dallas, TX). Cell-LightTM EdU Apollo®567 In Vitro Imaging Kit was from RiboBio (Guangzhou, China). Lactate assay kit was from Jiancheng Biotech (Nanjing, China). MG132, cycloheximide (CHX) and SU6656 were from MedChem Express (Monmouth Junction, NJ).
10
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blotting analysis, the proteins were extracted from modified cells with a protein lysis buffer containing 1 M Tris-Cl (pH 7.4), 1% (v/v) NP-40, 3 M NaCl, 0.5 M EDTA, 1% (v/v) TritonX-100 and 0.25 % (m/v) sodium deoxycholate. Western blotting was performed using primary antibodies: KMT2D (Invitrogen, PA5-49581), Bcl2 (Santa Cruz), Bax (Santa Cruz), CDH1 (Service), EpCAM (Service), CDH2 (Service), vimentin (Service), LIFR (MultiSciences), KLF4 (Biovision), H3 (Abcam), H3K4me1 (Abcam), ASH2L (Santa Cruz), RBBP5 (Biovision), WDR5 (Biovision) and GAPDH (Santa Cruz).
For the immunoprecipitation assays, whole-cell lysates were prepared as described above. First, 40 µL of protein A-Agarose (Santa Cruz) was added for 2 h at 4 °C. The input sample was 150 µL of supernatant, and the remainder was used for the experimental groups. KMT2D antibody (Atlas Antibodies, HPA035977) or isotype control (normal rabbit IgG, Beyotine) was added, and the mixture was incubated at 4 °C overnight. Then, the pellet was harvested and washed three times with the protein lysis buffer and stored at −80 °C for further analysis.
For the immunoprecipitation assays, whole-cell lysates were prepared as described above. First, 40 µL of protein A-Agarose (Santa Cruz) was added for 2 h at 4 °C. The input sample was 150 µL of supernatant, and the remainder was used for the experimental groups. KMT2D antibody (Atlas Antibodies, HPA035977) or isotype control (normal rabbit IgG, Beyotine) was added, and the mixture was incubated at 4 °C overnight. Then, the pellet was harvested and washed three times with the protein lysis buffer and stored at −80 °C for further analysis.
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!