Starch Assay Kit (ab83393)

Reviews (1)Q&A (6)References (4)

製品の概要

  • 製品名

    Starch Assay Kit

  • 検出方法

    Colorimetric/Fluorometric

  • サンプルの種類

    Cell Lysate, Tissue Lysate, Food samples

  • アッセイタイプ

    Quantitative

  • 検出感度

    > 4 µg/ml

  • 検出範囲

    4 µg/ml - 2000 µg/ml

  • 全工程の試験時間

    0h 40m

  • 製品の概要

    Abcam's Starch Assay Kit provides an easy, accurate assay to measure starch levels in a variety of samples. In the assay, starch is hydrolyzed to glucose which is oxidized to generate color (570 nm) and fluorescence (Ex/Em = 535/587 nm). The assay can detect starch at 0.0004 to 2 mg/ml.
    Visit our FAQs page for tips and troubleshooting.

  • 特記事項

    Starch is a complex carbohydrate consisting of a large number of glucose units. All plants contain starch, present as amylose, (linear α-1,4 linked polymer) and amylopectin, (highly α-1,6 branched α-1,4 polymer). Starch generally contains 0-25% amylose and 75-100% amylopectin.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • 試験プラットフォーム

    Microplate reader

製品の特性

画像

  • Functional Studies - Starch Assay Kit (ab83393)
    Functional Studies - Starch Assay Kit (ab83393)
    Starch Standard Curve: Different types of pure starch were extracted with 10N KOH/H3PO4 as described. Assays were performed following the kit protocol.

参考文献

ab83393 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab83393 は 4 報の論文で使用されています。

  • Song Y  et al. Regulation of Carbon Partitioning in the Seed of the Model Legume Medicago truncatula and Medicago orbicularis: A Comparative Approach. Front Plant Sci 8:2070 (2017). Functional Studies . PubMed: 29312368
  • Castro PH  et al. SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signaling and Metabolism in Arabidopsis thaliana. Plant Cell Physiol 56:2297-311 (2015). PubMed: 26468507
  • Mittal A  et al. TOPOISOMERASE 6B is involved in chromatin remodelling associated with control of carbon partitioning into secondary metabolites and cell walls, and epidermal morphogenesis in Arabidopsis. J Exp Bot N/A:N/A (2014). Functional Studies . PubMed: 24821950
  • Martin AR & Thomas SC Size-dependent changes in leaf and wood chemical traits in two Caribbean rainforest trees. Tree Physiol 33:1338-53 (2013). PubMed: 24336517

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1-7 of 7 Abreviews or Q&A

Question


Our customer has responded:

‘All in all, I have not detected enough amount of starch from both the soluble and insoluble supernatants.



So, I think I’m having issues with the starch extraction protocol. The starch is difficult to isolate by following the protocol.



I originally ground the seed tissues (I used 1-2 seeds roughly 10mg, normally 40% dry weight) with a plastic pestle in a 1.5 eppendorf tube (I’ve also tried sonificating the seeds) and added 1ml 90% ethanol washing twice to remove the glucose type sugar.

Then added 1ml MilliQ water and put it in the heating block (100degrees for 5mins), and then centrifuged 10k speed, take up the supernatant as soluble starch; continue to use 1ml 10M KOH to extract resistant starch, and sitting on heating block (100degrees for 5mins again), till cooled down and centrifuge to get the supernatant.



So far, I can get nothing or very little starch when I take the readings, and the reaction does not show the pink color that the standard starch is showing.

When extracting resistant starch, after the neutralization process, there are lots of salts. Is this normal?

I have checked the debris after resistant starch extraction, and there is a gel like layer? Does this mean that the starch is not soluble completely?

Do you have any other suggestions on how I can improve the extraction of soluble and insoluble starch from my seeds?’

Kind regards,

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Answer

Thank you for your response.

My colleague is out of office for a couple of days this week and she has asked me to look after her customers during her absence.

I am very sorry that your customer is having a hard time extracting starch from the seeds. Since we have not used this kit with seeds in-house, I can only make some suggestions as to what may help.



1) Longer incubation (more than 5 min) on the heating block might help.

2) Use more seeds initially (like 20 mg) to start seeing the pink colour development.


3) Increase the time of incubation in step 5, as I suggested in my previous email.



I can't comment on the salt concentration as that might be seed specific issue.



Hope this information has been helpful for you. Please let me know if you have any other questions.



Thanks,

Read More

Ab83393 - Starch Assay Kit for the analysis of fecal samples

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
Fecal samples were obtained by freeze-drying fresh feces.
Total Starch Quantification:
Extraction:
a)5-10 mg of ground fecal sample were weighted any free glucose and small oligosaccharides
were washed out with 1 ml 90% ethanol, warm to 60°C for 5 minutes with occasional vortexing (Eppendorf ThermoMixer was used).
b) It was centrifuged at 10,000 x g for 2 minutes.
c) the supernatant was discarded. Repeat the wash twice.
After the 90% ethanol wash, the washed sample was extracted directly with 10N KOH/H3PO4:
a) First, 1 ml 10N KOH was added, it was mixed with a vortex mixer and heated 5 minutes in Eppendorf ThermoMixer at 100°C. Let it cool down.
b) 10 N H3PO4 was added slowly dropwise. Around 0,55 mL of the acid was needed for the neutralization. The pH was checked with a litmus test.
c) Samples were centrifuged at 10,000 x g for 2 minutes. The supernatant was moved into a clean tube and centrifuged again at 10,000 x g for 2 minutes (photo2). An "upper residue" in addition to the pellet was seen. The clear supernatant was sampled with a needle (syringe), in order not to take the insoluble residue that did not precipitate.
Plate preparation:
20 μl of the extracted starch in fecal samples was taken, 180 μl of Hydrolysis Buffer was addend end mixed.
Up to 50 μl of the diluted sample (in this case 40) or buffer (blank) was added to test wells.
The volume of test well were adjusted to 50 μl with Hydrolysis Buffer.
Standard Curve Preparation: it was prepared as in the protocol.
Hydrolysis*:
Hydrolysis Enzyme Mix 2 μl was added and mixed well. Incubate for at least 30 minutes at room temperature to hydrolyze starch.
Development: For each well, a total 50 μl of Reaction Mix was prepared and added to each well, and let incubate at room temperature for 30 minutes, protect from light.
5. Measurement: the plate was measured colorimetrically (OD560nm) with Glomax (Promega)

Elena Dalle Vedove

Verified customer

投稿 Mar 11 2020

Question

Hi,

The customer has responded regarding this starch assay kit:

;Now the measurement of resistant starch seemed to work alright and I 've checked it against a few plant species. The recent trials, I used DMSO rather than 10M KOH (neutralization by H3PO4) to dissolve the resistant starch. I just wish to provide good feedback to you in terms of the kit use for plant seeds if others may use. The kit is easy to use and very useful for quick measurement. Thanks’

Kind regards,

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Answer

Thank you very much for that feedback. This sort of information is indeed very important for your customers.

I would strongly encourage your customer to provide us with an Abreview for the product. This would simply involve sharing the protocol details of the experiment and some data obtained which I would then add to the datasheet of the product.

More information on the Abreview system can be found here:

https://www.abcam.com/Abreview

As a reward your customer would receive Abpoints which can go towards a future Abcam purchase, or can be claimed as Amazon vouchers. Information on the Abpoints can be found from the following link:

https://www.abcam.com/abpoints

I hope this information has been of help. If your customer would like to submit the Abreview they will need to do it directly to me as we are currently unable to support Abreviews for kits online. We are hoping to remedy this soon.

Please do pass on our thanks to the customer and I hope they will consider submitting an Abreview.

Have a nice weekend.

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Question

Our customer has responded to your questions:

1. Have you performed the standard curve? Has this produced the expected graph? What were your readings?

I performed a standard curve, but I am not sure whether this has produced the expected graph. The data for my standard curve were as follows:

Starch quantity ug

OD value at 570 nm

######

2. Could you describe exactly how the samples were obtained, extracted and washed?

The seeds were isolated and kept in a -80 freezer, and ground with a plastic pestle and mortar and washed with 90% ethanol.


3. Was a glucose control performed (not using hydrolysis enzyme)? What were the result of this reading?

I did, but I used it as blank (readings were measured via spectrophotometer)

4. You say that you see erratic readings "it changes a lot every few minutes", does it just go up, or up and down? What values are you seeing?

The reaction seemed not to be finished after 30 mins. When I read the OD value every few minutes, the OD value was not stable, it always went up in 1-2 min intervals. Should the reading output be significantly affected by the specific time that you measure samples after the incubation period (E.g. if there is a 10-minute period between the first and last samples being read, will readings still be accurate?)?

5. Are the values seen within the expected detection limit of the kit (0.0004-2 mg/mL)?

Not sure, I have not calculated, this should not be a problem.

6. Were the buffers used allowed to come to room temperature prior to carrying out the protocol?

Yes

Thanks very much for your help.

Kind regards,

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Answer

Thank you once again for your enquiries which have been forwarded to me as my colleague Karin is currently away from the office.

Karin has discussed this withour source for this antibody, and they have kindly provided the following advise:

1. All the details described in the protocol on the datasheet are standardized for samples different from these seeds. In order to get comparable results with these seeds, some tweaking of the details/optimization has to be performed by the end user. So the 30 min incubation time after which the absorbance is still increasing, might indicate that this time has to be standardized for these samples. I would suggest a time course experiment with times ranging upwards of 30 min, to see the optimal end point time.

What was the maximum time at which the OD was measured?

2. I agree the standard readings are very low. It may be due to various reasons:

- Improperly thawed components - Thaw all components completely and mix gently before use

- Use of expired kit or improperly stored reagents - Always check the expiry date and store the components appropriately

- Allowing the reagents to sit for extended times on ice - Always thaw and prepare fresh reaction mix before use

- Incorrect incubation times or temperatures - Refer datasheet & verify correct incubation times and temperatures

- Incorrect volumes used.

It might also happen if the OD is not measured at exactly 570 nm.

I hope this information will be helpful and help optimize the results from the sample. However, I am concerned that. Please let me know how the customer gets on and how they would like to proceed in this case.

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Question

Hi,

A researcher has contacted us in regards to this product: Starch Assay [Ab83393]

“ I am using the starch assay kit to measure soluble starch levels in Medicago seeds. I am measuring the starch content, via the colorimetric method (read via spectrophotometer).

After the 30-minute incubation, the OD values are increasing the whole time. Is the reaction still continuing after the 30-min incubation?

I am also finding that the OD value is not consistent each time we measure the data. When I read the OD values, it changes alot every few minutes. Could you please provide some trouble-shooting advice?”

Kind regards,

Read More
Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to understand more fully what the problem may be could I please ask you to answer the following questions:

1. Have you performed the standard curve? Has this produced the expected graph?

2. Could you describe exactly how the samples were obtained, extracted and washed?

3. Was a glucose control performed (not using hydrolysis enzyme)? What were the result of this reading?

4. You say that you see erratic readings "it changes a lot every few minutes", does it just go up, or up and down? What values are you seeing?

5. Are the values seen within the expected detection limit of the kit (0.0004-2 mg/mL)?

6. Were the buffers used allowed to come to room temperature prior to carrying out the protocol?

With this information I will hopefully understand more fully what may be contributing to the problems you have been observing.

I look forward to receiving your reply.

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Question

Our customer would like to inquire about the Starch Assay Kit (ab83393). The sample he would like to test is the juveniles and leaves of Arabidopsis thaliana, but he is not sure whether ab83393 is suitable. And if it is suitable for Arabidopsis thaliana, could you please offer the suggestive starch extraction protocol for Arabidopsis thaliana? Would you please help him provide the information? Thank you very much.

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Answer

Thank you for contacting us.

Theoretically this kit can be used with any type of starch containing samples. I assume it can detect Starch inleaves of Arabidopsis thaliana also. Please be advised that we haven't used this kit for detection of starch inleaves of Arabidopsis thalianaso we will not be able to provide any tested data. The optimal experimental conditions has to be determined by the end user.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

Is this https://www.abcam.com/Starch-Assay-Kit-ab83393.html suitable to use for plant (legume) seeds?



Kind regards,

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Answer

Thank you for contacting us.

Theoretically this kit can be used with any type of starch containing samples. I assume it can detect Starch in legume seeds also. please be advised that we haven't used this kit for detection of starch in legume seeds so we will not be able to provide any tested data. The optimal experimental conditions has to be determined by the end user.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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