Proteomics workflow for whole cell lysate, endosome, and lysosome fractions v1

2021 ◽  
Author(s):  
Hankum Park ◽  
Frances V Hundley ◽  
J. Wade Harper
Keyword(s):  
Sample Preparation ◽  
Whole Cell ◽  
Cell Lysates ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Ms Analysis

We present a protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP. Protocols for purification of lysosomes and endosomes is provided in protocol dx.doi.org/10.17504/protocols.io.byi9puh6 using cells that express endogenously tagged TMEM192-HA and stably expressing FLAG-EEA1 as descrbed in dx.doi.org/10.17504/protocols.io.byi7puhn.

Proteomics workflow for whole cell lysate, endosome, and lysosome fractions v2

2021 ◽  
Author(s):  
Hankum Park ◽  
Frances V Hundley ◽  
J. Wade Harper
Keyword(s):  
Sample Preparation ◽  
Whole Cell ◽  
Cell Lysates ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Ms Analysis

We present a protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP. Protocols for purification of lysosomes and endosomes is provided in protocol dx.doi.org/10.17504/protocols.io.byi9puh6 using cells that express endogenously tagged TMEM192-HA and stably expressing FLAG-EEA1 as descrbed in dx.doi.org/10.17504/protocols.io.byi7puhn.

scholarly journals Nano-LC FTICR Tandem Mass Spectrometry for Top-Down Proteomics: Routine Baseline Unit Mass Resolution of Whole Cell Lysate Proteins up to 72 kDa

2012 ◽  
Vol 84 (5) ◽  
pp. 2111-2117 ◽  
Author(s):  
Jeremiah D. Tipton ◽  
John C. Tran ◽  
Adam D. Catherman ◽  
Dorothy R. Ahlf ◽  
Kenneth R. Durbin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Mass Resolution ◽  
Unit Mass ◽  
Tandem Mass ◽  
Top Down ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Unit Mass Resolution

scholarly journals Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0131364 ◽  
Author(s):  
Iris Bosschem ◽  
Jagadeesh Bayry ◽  
Ellen De Bruyne ◽  
Kim Van Deun ◽  
Annemieke Smet ◽  
...  
Keyword(s):  
Side Effects ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate

Initiation by RNA polymerase II and formation of runoff transcripts containing unblocked and unmethylated 5' termini

1983 ◽  
Vol 3 (12) ◽  
pp. 2172-2179
Author(s):  
H Ernst ◽  
W Filipowicz ◽  
A J Shatkin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Atp Hydrolysis ◽  
Beta Globin ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Promoter Dna ◽  
Gamma S ◽  
Made In

Transcription of cloned adenovirus, beta-globin, and retrovirus long terminal repeat DNAs in HeLa whole-cell lysate was inhibited by S-adenosylhomocysteine. However, full-length 1.7-kilobase transcripts made on adenovirus 2 late promoter DNA contained 5'-terminal GpppA, consistent with specific initiation and runoff synthesis in the absence of product methylation. Formation of runoff transcripts including retrovirus RNAs that normally contain 5'-m7GpppGmpC was not decreased by replacing GTP with non-hydrolyzable analogs, and Rous-associated virus-2 runoff products made in the presence of GTP-gamma-S contained 5'-terminal gamma-S-pppGpC. The results indicate that capping and specific transcript synthesis by RNA polymerase II are not obligatorily linked in HeLa whole-cell lysate. Accurate initiation is dependent on ATP hydrolysis, and in contrast to GTP, replacement of ATP by 5'-adenylyl-imidodiphosphate blocked specific initiation of transcripts that start with either GTP (Rous-associated virus-2, Rous-associated virus-0) or ATP (beta-globin, adenovirus).

Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids

2015 ◽  
Vol 60 (4) ◽  
Author(s):  
Jaideep Kumar ◽  
Ashok Chaudhury ◽  
Bidhan C. Bera ◽  
Ritesh Kumar ◽  
Rajender Kumar ◽  
...  
Keyword(s):  
Ethical Issues ◽  
Terminal Region ◽  
Antibody Detection ◽  
Preliminary Evaluation ◽  
Large Set ◽  
In Vitro Production ◽  
Serum Samples ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate

AbstractThe present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress

2016 ◽  
Vol 200 ◽  
pp. 90-101 ◽  
Author(s):  
Amenehsadat Hashemi ◽  
Javad Gharechahi ◽  
Ghorbanali Nematzadeh ◽  
Faezeh Shekari ◽  
Seyed Abdollah Hosseini ◽  
...  
Keyword(s):  
Salt Stress ◽  
Protein Complexes ◽  
Two Dimensional ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Sds Page ◽  
Blue Native

scholarly journals ESCORTing proteins directly from whole cell-lysate for single-molecule studies

2017 ◽  
Vol 535 ◽  
pp. 35-42 ◽  
Author(s):  
Shwetha Srinivasan ◽  
Jagadish P. Hazra ◽  
Gayathri S. Singaraju ◽  
Debadutta Deb ◽  
Sabyasachi Rakshit
Keyword(s):  
Single Molecule ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Single Molecule Studies

scholarly journals Identification of immunodominant Helicobacter pylori proteins with reactivity to H. pylori-specific egg-yolk immunoglobulin

2003 ◽  
Vol 52 (3) ◽  
pp. 217-222 ◽  
Author(s):  
Ji-Hyun Shin ◽  
Seung-Woo Nam ◽  
Jung-Taik Kim ◽  
Jong-Bok Yoon ◽  
Won-Gi Bang ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Egg Yolk ◽  
Cross Reactivity ◽  
Heat Shock Protein 60 ◽  
Α Subunit ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Normal Human ◽  
H Pylori

The importance of hens eggs as a source of specific antibodies (IgY) is well recognized. The protective effect of IgY obtained from hens immunized with Helicobacter pylori whole-cell lysate has been reported for the control of H. pylori infection. However, IgY produced by whole-cell lysates presents the possibility of cross-reactivity with other bacteria, including the normal human flora, and this could decrease the efficiency of IgY. In the present study, the immunodominant proteins of H. pylori with reactivity to H. pylori-specific IgY (IgY-Hp) were identified. IgY obtained from hens immunized with various fractions of H. pylori proteins was isolated and purified, titres of IgY-Hp against H. pylori were determined and cross-reactivity between IgY-Hp and normal human bacteria was examined by Western blot analysis. Finally, immunodominant H. pylori proteins were identified by LC/MS analysis. IgY obtained 2 months after immunization with H. pylori whole-cell lysate showed the highest antibody titre. Five immunodominant proteins were identified that were strongly reactive to IgY-Hp: urease β-subunit (62 kDa), heat-shock protein 60 (60 kDa), urease α-subunit (26 kDa), probable peroxiredoxin (22 kDa) and probable thiol peroxidase (18 kDa). Immunization of hens with the immunodominant proteins identified would produce a more specific IgY against H. pylori.

scholarly journals A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

2012 ◽  
Vol 2 (1) ◽  
pp. 5 ◽  
Author(s):  
James DR Knight ◽  
Ruijun Tian ◽  
Robin EC Lee ◽  
Fangjun Wang ◽  
Ariane Beauvais ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Kinase Assay ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate
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