Tag Archives: protein

Using ESearch and EFetch to retrieve data

Lets say there was this person, we will call her Emma for now, that needed to download lots of data but wanted to make it more robust and reliable. Here is a way to use NCBI ESearch and EFetch tools to do so. Complete documention at http://www.ncbi.nlm.nih.gov/books/NBK25498/. Specific example used is here


Example one: Download all ilumatobacter protein sequences in fasta format.

Will use Esearch to get GI numbers, post them to history and multiple EFetch calls to retrieve data.

Input: $query – ilumatobacter[orgn]

Output: A file named “ilumatobacter.fa” containing FASTA data.

Perl script


use LWP::Simple;
$query = 'ilumatobacter[orgn]';

#assemble the esearch URL
$base = 'http://eutils.ncbi.nlm.nih.gov/entrez/eutils/';
$url = $base . "esearch.fcgi?db=protein&term=$query&usehistory=y";

#post the esearch URL
$output = get($url);

#parse WebEnv, QueryKey and Count (# records retrieved)
$web = $1 if ($output =~ /(S+)</WebEnv>/);
$key = $1 if ($output =~ /(d+)</QueryKey>/);
$count = $1 if ($output =~ /(d+)</Count>/);

#open output file for writing
open(OUT, ">ilumatobacter.fa") || die "Can't open file!n";

#retrieve data in batches of 500
$retmax = 500;
for ($retstart = 0; $retstart < $count; $retstart += $retmax) {
$efetch_url = $base ."efetch.fcgi?db=protein&WebEnv=$web";
$efetch_url .= "&query_key=$key&retstart=$retstart";
$efetch_url .= "&retmax=$retmax&rettype=fasta&retmode=text";
$efetch_out = get($efetch_url);
print OUT "$efetch_out";
}
close OUT;

So if you wanted to use this simple paste the above code in text file (Suggest using TextWrangler) and saving as .pl file (ie /Users/sr320/Desktop/ill-prot.pl. Then in Terminal, type perl /Users/sr320/Desktop/ill-prot.pl. The data will download to whatever directory you are in Terminal.

In actuallity, this still seems to fail randomly. This is common to see on the internets. The best guess is too many requests during busy time of day, so it might take a couple if trys. See http://www.ncbi.nlm.nih.gov/books/NBK25497/#chapter2.Usage_Guidelines_and_Requiremen for usage recommendations.

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SDS/PAGE, Western Blot – Test of new Western Breeze Kit & HSP70 Ab for FISH441

Ran varying amounts of Gigas Gill 24hr Control protein extract from 20080917. See table below:

Protein (ug) Volume of Sample (uL) Water up to 15uL (uL)
5 1.56 13.44
10 3.125 11.88
15 4.69 10.31
20 6.25 8.75
30 9.38 5.63
40 12.5 2.5
50 15.65

Volumes were adjusted to 15uL with H2O and combined with 15uL of 2X Reducing Sample Buffer in screw cap tubes. Also, 15uL of MSTN was mixed with 15uL of 2X Reducing Sample Buffer in a screw cap tube. This will serve as a positive control for the kit. Samples were boiled for 5mins. and then microfuged for 1min @ max speed @ 4C. Samples were loaded on Pierce 4-20% Tris-HEPES SDS PAGE gels and run 150V for 45mins. 10uL of SeeBlue Plus ladder and 10uL of the 12-tag positive control lysate were also loaded on the gel.

external image 20081231.JPG

Gel, membrane, and blotting paper were soaked for 15mins in transfer buffer (Pierce recipe w/methanol). Proteins were transferred to nitrocellulose membrane for 30mins @ 20V. Gel was stained for ~40mins in Coomassie stain and then destained w/10% acetic acid solution until bands were clearly visible.

After transfer, membrane was cut into two (one with the gigas samples and the other with the MSTN and the 12-tag lysate samples) and developed separately. Western blotting was done according to Invitrogen WesternBreeze Chromogenic (anti-mouse) protocol. Primary anti-HSP70 Ab was used at a 1:5000 dilution (2uL) and the primary anti-myc tag Ab (for MSTN) was used at a 1:1000 dilution (10uL). The anti-myc blot was allowed to develop for ~1min before stopping. The anti-HSP70 blot was allowed to develop for ~1.5hrs before stopping.

external image 20081231-01.JPGexternal image 20081231-02.JPG

Results:

 

The anti-HSP70 blot showed no bands. The anti-myc blot showed MSTN1b and positive control lysate bands. This confirms that the kit works properly.

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SDS/PAGE/Western – anti-HSP70 Ab Re-test

Another attempt to determine appropriate amounts of anti-HSP70 Ab (ABR cat# MA3-006)and/or protein needed for better detection of HSP70 in Gigas protein samples. Gigas gill protein extracts from 20080617 (both control and Vibrio exposure samples) were each pooled. The two samples were mixed with an equal volume of 2x sample reducing buffer. 100uL of hemolymph were extracted from Gigas muscles and mixed with an equal volume of 2x sample reducing buffer. Samples were boiled for 5mins. and loaded onto a Pierce 4-20% tris-hepes gel. Also loaded 10uL of SeeBlue ladder. Ran gel @ 150V for 45mins. Samples were transferred to nitrocellulose 20V for 30mins. Well locations were marked on the membrane with a pencil. Gel was stained with Coomassie stain for 30 mins and then destained with 10% acetic acid until bands were clearly visible.

external image 20081216.JPG

Lane 1 – Ladder
Lane 2 – Control (20ug)
Lane 3 – VE (20ug)
Lane 4 – Hemos (40uL)
Lane 5 – Control (10ug)
Lane 6 – VE (10ug)
Lane 7 – Hemos (20uL)

Lane 8 – Control (20ug)
Lane 9 – VE (20ug)
Lane 10 – Hemos (40uL)
Lane 11 – Control (10ug)
Lane 12 – VE (10ug)

Membrane was cut in two (between lanes 7 & 8) for probing with two different primary Ab concentrations: 1:5000 (per the manufacturer’s suggestion) and 1:2500. Membranes were incubated 1hr. in 15 mL of blocking solution. Primary Ab was added at the two above mentioned concentrations to the respective membrane and incubated 1hr. Membranes were washed with 1x TBS-T 3 x 10mins. Secondary Ab (DAM-HRP) was added with blocking solution to the membranes at a 1:2500 dilution and incubated 30mins. Membranes were washed with 1x TBS-T 3 x 10mins. Membranes were developed with Millipore Immobilon Western Chemiluminescent reagent and imaged together.

Results:

No image of any sort! Not even the pencil marks were visible. Just a blank screen. I’m starting to suspect that something is wrong with the imaging system or something. This is basically a repeat of the Western on 20081210 which worked. Now I’m not sure what to do at all. This blows.

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SDS/PAGE/Western – anti-HSP70 Ab test

Test of the new anti-HSP70 Ab (ABR cat# MA3-006). Gigas gill protein extracts from 20080617 (both control and Vibrio exposure samples) were each pooled to result in 10ug protein of control and 10ug protein of VE in a volume of 10uL each. The two samples were mixed with an equal volume of 2x sample reducing buffer. O. rubescans samples were taken from Rachel’s -20C box. 15uL of each sample was mixed with 2x sample reducing buffer. All samples were boiled for 5mins and spot spun. Samples were loaded onto Pierce 4-20% Tris-Glycine SDS/PAGE gels. 10uL of SeeBlue ladder was loaded. Gel was run @ 150V for 45mins.

Gel was transferred to nitrocellulose membrane (equilibrated in tris-glycine transfer buffer for 5mins) @15V for 20mins. Membrane washed briefly in 1x TBS-T, then blocked for 1hr. in 15mL of blocking solution. Primary Ab was added at a 1:5000 dilution per the HSP70 data sheet and incubated O/N @ 4C. Gel was stained for 1hr in Coomassie stain and then destained w/ 10% acetic acid O/N.

Lane #1 – Ladder

Lane #2 – Gigas gill control pool

Lane #3 – Gigas vibrio exposure pool

Lane #4 – O. rubescans “mucus from suction” 10/24

Lane #5 – O. rubescans “UW 11/21″

Lane #6 – O. rubescans “Bucket 11/21″

Lane #7 – O. rubescans “water/mucus from bucket 10/24″

Lane #8 – O. rubescans “skin 11/21″

Results: The two oyster samples are the only two that are visible on the coomassie stained gel. I will have to talk to Rachel concerning the columes of O. rubescans proteins that she loaded on her gels, as it is NOT clear in her notebook entries. Additionally, the gel did not destain very well.

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