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GlnA | glutamine synthetase positive control/quantitation standard

238 €

AS09 018S  |  recombinant protein standard for quantitation and positive control

Buy 2 positive controls/standards for the price of 1.
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Follow this link to a list of discounted standards.


PRODUCT INFORMATION IN PDF

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AS09 018S

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product information
Background

Glutamine synthetase (GlnA) is the key enzyme in the incorporation of mineral nitrogen into glutamine.

This product is a recombinant GlnA protein standard, source Synechocystis strain PCC 6803.

Host
Clonality
Clone
Purity
Format Lyophilized
Quantity 250 µl
Reconstitution For reconstitution add 225 µl of milliQ water
Storage

store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

Tested applications Western blot (WB)
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Plant and algal protein extraction buffer

Secondary antibodies

Additional information

The GlnA protein standard can be used in combination with global anti-GlnA antibodies to quantitate GlnA from a wide range of species. Global antibodies are raised against highly conserved amino acid sequences in the GlnA protein.

Quantitative western blot:  detailed method description, video tutorial

application information
Recommended dilution

Standard curve: 3 loads are recommended (0.5, 2 and 4μl).
For most applications a sample load of 0.2μg of chlorophyll will give a GlnA signal in this range.

Positive control: a 2μl load per well is optimal for most chemiluminescent detection systems. 

This standard is stabilized and ready and does not require heating before loading on the gel.

Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

Expected | apparent MW

in most gel systems GlnA migrates around 53 kDa

Confirmed reactivity
Predicted reactivity
Not reactive in

no confirmed exceptions from predicted reactivity known in the moment

Additional information

Concentration: after adding 225  µl of milliQ water final concentration of the standard is 0.20 pmol/µl

Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl 106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red 0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50mM DTT.

This standard is ready-to-load and does not require any additions or heating. It needs to be fully thawed and thoroughly mixed prior to using. Avoid vigorous vortexing, as buffers contain detergent. Following mixing, briefly pulse in a microcentrifuge to collect material from cap.
This standard is stabilized and ready and does not require heating before loading on the gel.
Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

Selected references

to be added when available


application example

3 µg of total protein from Trichodesmium IMS 101 extracted with PEB (AS08 300) (1-8) and GlnA protein standard 0.3, 0.15, 0.07 pmol (9-11) were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horseradish peroxidase conjugated, from Abcam) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according the manufacturer's instructions. Images of the blots were obtained using a CCD imager nd Quantity One software  Exposure time was 10 seconds.
Note: Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.

 

  western blot using anti- GlnA antibodies

Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.

Quantitative western blot:  detailed method description.


||| For other applications or usage on species other than stated above Agrisera offers a payment-after-testing option. To learn more about this or for any questions please use the LiveChat option or contact us at support@agrisera.com