10kD Spin Column (ab93349)

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Overview

  • Product name
    10kD Spin Column
  • Product overview

    The 10kD Spin Columns (ab93349) are disposable ultrafiltration devices for the concentration or deproteinization of biological samples.


    They can be used in either a swing bucket or fixed angle rotors which accept 2.0 mL centrifuge tube at maximum speed 10000 x g (5-10 mins). The vertical membrane design and thin channel filtration chamber minimizes membrane fouling and provides high speed concentration, even laden solutions. It is specifically designed with low internal surface and membrane area in order to achieve superior recovery from dilute solution.


    Maximum initial sample volume is 500µL. The dead stop volume is approximately 20-25µL.

  • Notes

    If working with cell or tissue lysates, we generally recommend the TCA Sample Deproteinization Kit or using a PCA/KOH Deproteinization protocol. If it is critical to use a spin column with a cell or tissue lysate then, to avoid blocking the filter, first centrifuge the lysate samples at ~14,000 rpm for 10-20 mins to pellet debris, and then use the resulting supernatant with the spin column.

  • Tested applications
    Suitable for: Purificationmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab93349 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Purification Use at an assay dependent concentration.

Images

  • - 10kD Spin Column (ab93349)
    - 10kD Spin Column (ab93349)

References

This product has been referenced in:
  • Rao P & Suvas S Development of Inflammatory Hypoxia and Prevalence of Glycolytic Metabolism in Progressing Herpes Stromal Keratitis Lesions. J Immunol 202:514-526 (2019). Read more (PubMed: 30530484) »
  • Stout AC  et al. Inflammation and glucose homeostasis are associated with specific structural features among adults without knee osteoarthritis: a cross-sectional study from the osteoarthritis initiative. BMC Musculoskelet Disord 19:1 (2018). Read more (PubMed: 29304778) »
See all 14 Publications for this product

Publishing research using ab93349? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question

I'm using the NAD assay kit and I just purchased the 10kDa cutoff filters. I'm just wondering how long do you recommend spinning at 10,000 g to deproteinize 400ul of lysates ? Thanks

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Answer



It is hard to predict how long will be required to spin the volume through the spin filter. It depends on how much protein is in the solution to be filtered as higher protein concentrations require longer spin times. I recommend spinning for 5 min at 4 deg C at first and then check to see how much volume has gone through. Then you will have a good idea as to how much longer will be required to push the remainder through.

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Question

using plasma i can only get about 100 ul of my 500 ul sample to go through, even after centrifuging multiple times. how can i improve this, or is there some problem with the columns?

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Answer

Thank you for contacting us.

Always ensure that you do not load more than 450 µl of diluted, non-viscous samples. I understand in the case of plasma you may not wish to dilute further. If you still have a difficulty in recovering a reasonable volume back, please centrifuge the samples for ˜30-45 min at 12000 rpm at 4°C.

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





ein Frage zu den 10 kD spin columns (ab93349). Bei der Verwendung im Anschluss an die Zellyse mit ATP assay buffer für den ATP assay kit , verwendet man nach der Zentrifugation den Durchfluss oder Überstand oberhalb der Membran für den assay?



Ich

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Answer



Ich kann bestätigen, dass die "spin cloumns" verwendet werden um alles, das grösser ist als 10kDa loszuwerden.

Bitte verwenden Sie den deshalb Durchfluss im ATP assay kit.

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Question

How have these columns been tested to ensure that all 10 kDa proteins are removed and >10 kDa proteins are retained?

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Answer



The columns have been tested to ensure that the samples are separated correctly. The fragments have been tested using several of our assay kits, verifying that analytes above/below 10 kDa are present in the correct fragment.

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Question

I am using the 10kDa columns in preparing samples for a Lactate Assay. I collected the cells from the culture plate and passed them through a needle prior to adding the lysate onto the columns. When I spin down the columns at 10000rpm for 5 mins, I get about 50uL elute from 500uL input. I still see the majority of the lysate solution within the column and a small clump of solid at the bottom (likely the cellular debris). I wanted to know if all of the lysate input should be absorbed by the filter or if it is normal for most of it to remain in the column? Could the cell debris be blocking the column? Also, once the elute has come through, do I need to spec it to make sure I use equal amounts of protein concentrations in my assay, or do I assume I get the same amount from the columns?

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Answer

You are exactly correct, insoluble material, whether from cells or tissue, needs to be removed prior to loading into the 10kDa spin column. Thus, after lysis/homogenization, centrifuge your sample to pellet the insoluble material. Then take the supernatant as your sample and load into the 10kDa spin column.

Typically, an equivalent volume of sample will be loaded per well as the assay measures the amount of lactate present, but please refer to your lactate assay kit protocol. One of our lactate assay protocols is provided through the link below and uses an equivalent volume of sample per well:
https://www.abcam.com/ps/products/65/ab65331/documents/ab65331%20L%20Lactate%20Assay%20Kit%20Colorimetric%20(Website).pdf

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Question

Froze cells at -80C prior to lysis. Following lysis, if 10kDa filter is not used, is it ok to freeze samples? She wants to avoid using the 10kDa filter if possible and will run controls to determine if it causes any variability.

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Answer

We recommend the use of the 10 kDa filters irrespective of your immediate or later usage of the lysed samples. The filters help in separating out proteins which can chew up the analyte. You most likely will not get efficient results without the fliters.

We carry 10 kD spin columns to assist in using this kit. The link for these columns is given below:
https://www.abcam.com/10kd-spin-column-ab93349.html

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Question

Thanks for your answer.
My concern was that using the assay buffer to homogenize the samples might generate a lot of bubbles (if the buffer has any kind of detergent).
Can I extract lactate from samples using ethanol 80% and remove the proteins with perchloric acid?

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Answer

Thanks for your response.
I strongly recommend sticking to the kit protocol. Adding solutions (such as ethanol and perchloric acid) other than those provided in the kit will compromise the efficiency of lactate detection. The centrifugation step can be used to remove bubbles after homogenization in the assay buffer.
I hope this response proves helpful. Please do not hesitate to reply or contact us with any additional questions.

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Question

Product code: 65330
Lot number: XXXXXX
º
Inquiry: Hi, We just bought the L-Lactate assay kit and I cannot find in the assay protocol how should I homogenize my tissue samples (hypothalamus). I will apreciate information about this issue in order to process the samples. Thanks

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Answer

Thank you for contacting Abcam.
Since the lactate dehydrogenase (LDH) in tissue lysates degrades lactate, be sure to keep your samples at -80ºC. Below is our recommended homogenization protocol:
1. Homogenize the tissue samples (should contain at least 2 million cells) in the assay buffer that is provided for 10-50 passes on ice.
2. Centrifuge the sample and collect the supernatant.
3. Load the supernatant onto a 10kda spin column (ab93349 https://www.abcam.com/10kd-spin-column-ab93349.html) for deproteinization to remove LDH. Use the eluate for your subsequent assays.
To make sure that your readings from these samples are within the standard curve, we suggest using several doses of your sample when running your assay.
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

Inquiry: Hello, I was wondering if you could please recommend me a product. I need to extract water-soluble metabolites from cell lysate in order to analyse them using a HPLC. So I'm looking for a microcentrifuge spin-through filter that will get rid of all the proteins and only yield the metabolites as a flow-through. Could you please tell me if I can use the 10 kD spin columns (ab93349) for this purpose? Or if I should get a filter with smaller pores such as a 3 kD filter? Many thanks,

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Answer

Thank you for contacting us.

Yes, you can definitely use 10 kD spin filter to filter out all the proteins from samples. We routinely use and recommend these filters in conjugation with many of our assays kits.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

Protocole pour ab93349?

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Answer

Merci de nous avoir contactés.

Nous suggérons de mettre votre surnageant (maximum 500 ul) dans la colonne à une vitesse de 10000 x g pour 5 minutes. Par contre les temps varient donc il se peut que 5 minutes ne suffisent pas et qu’une centrifugation plus longue soit nécessaire.

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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