CDK12 Antibody (Rabbit mAb) [N24C20]

Catalog No.: F7665

Application: Reactivity: Human, Mouse, Rat

Lane 1: Hela, Lane 2: 293T, Lane 3: K-562, Lane 4: NIH/3T3

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

キーポイント

WB
SDS-PAGE の分離ゲルの推奨濃度：5%。
推奨WB希釈率: 1:10000

使用情報

Dilution
WB1:10000 IP1:30 IHC1:500

Application
WB, IP, IHC

Source
Rabbit Monoclonal Antibody

Reactivity
Human, Mouse, Rat

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
164 kDa 205 kDa,124 kDa
^*なぜ予測分子量と実際の分子量が異なるのか？下記の原因により、実際の分子量が予測と異なる：タンパク質の翻訳後修飾（リン酸化/糖鎖付加），スプライシングバリアント，イソフォーム，相対的な電荷，ポリマー。

ポジティブコントロール	Human colon carcinoma tissue; Rat colon tissue; Mouse cerebrum tissue; Mouse colon tissue; Human stomach tissue; Human testis tissue; Rat testis tissue; Mouse testis tissue; HeLa cells; 293T cells; K-562 cells; NIH/3T3 cells; PC-12 cells
ネガティブコントロール

プロトコール

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

生物学的記述

Specificity
CDK12 Antibody (Rabbit mAb) [N24C20] detects endogenous levels of total CDK12 protein.

タンパク質の局在
細胞核

Uniprot ID
Q9NYV4

Clone
N24C20

Synonym(s)
CRK7, CRKRS, KIAA0904, CDK12, Cyclin-dependent kinase 12, Cell division cycle 2-related protein kinase 7, Cell division protein kinase 12, CrkRS, CDC2-related protein kinase 7, Hcdk12

Background
CDK12 is a transcription‑associated cyclin‑dependent kinase that forms an active complex with cyclin K and functions as a carboxyl‑terminal domain (CTD) kinase for RNA polymerase II, thereby acting as a global regulator of transcription elongation and RNA processing with particular impact on long genes involved in DNA damage responses and genome maintenance. The kinase contains a canonical CDK catalytic core with an activation loop and a large C‑terminal extension that contributes to CTD binding and substrate selectivity, and the CDK12–cyclin K complex phosphorylates Ser2 and Ser5 residues within heptad repeats of the RNAPII CTD, enhancing processive elongation and productive transcription across gene bodies. CDK12 activity stimulates RNAPII elongation on a broad set of protein‑coding genes and also influences transcription termination, co‑transcriptional splicing, and RNA turnover, in part through direct phosphorylation of RNA‑processing factors that associate with the elongating polymerase. CDK12 has a strong functional bias toward long, complex genes encoding key DNA damage response and homologous recombination proteins such as BRCA1, ATR, FANCI, and other HR factors; inhibition or loss of CDK12 causes gene length‑dependent elongation defects, premature cleavage and polyadenylation within introns, and marked down‑regulation of these DDR transcripts, leading to reduced DNA repair capacity and genomic instability. This selectivity arises from the combination of CTD phosphorylation defects and altered regulation of pre‑mRNA processing at intronic polyadenylation sites, making long DDR genes particularly sensitive to reduced CDK12‑dependent elongation and processing. CDK12 also regulates expression of core DNA replication genes and other cell‑cycle regulators by controlling RNAPII processivity over these loci, thereby contributing to proper G1/S progression and coordination between replication and repair. In cancer, recurrent CDK12 loss‑of‑function mutations occur in high‑grade serous ovarian carcinoma, prostate cancer, and other tumor types and associate with tandem duplications, focal genomic rearrangements, and a distinct genomic instability signature that reflects defective transcription of homologous recombination genes rather than direct catalytic lesions in replication or repair enzymes.

Background

CDK12 is a transcription‑associated cyclin‑dependent kinase that forms an active complex with cyclin K and functions as a carboxyl‑terminal domain (CTD) kinase for RNA polymerase II, thereby acting as a global regulator of transcription elongation and RNA processing with particular impact on long genes involved in DNA damage responses and genome maintenance. The kinase contains a canonical CDK catalytic core with an activation loop and a large C‑terminal extension that contributes to CTD binding and substrate selectivity, and the CDK12–cyclin K complex phosphorylates Ser2 and Ser5 residues within heptad repeats of the RNAPII CTD, enhancing processive elongation and productive transcription across gene bodies. CDK12 activity stimulates RNAPII elongation on a broad set of protein‑coding genes and also influences transcription termination, co‑transcriptional splicing, and RNA turnover, in part through direct phosphorylation of RNA‑processing factors that associate with the elongating polymerase. CDK12 has a strong functional bias toward long, complex genes encoding key DNA damage response and homologous recombination proteins such as BRCA1, ATR, FANCI, and other HR factors; inhibition or loss of CDK12 causes gene length‑dependent elongation defects, premature cleavage and polyadenylation within introns, and marked down‑regulation of these DDR transcripts, leading to reduced DNA repair capacity and genomic instability. This selectivity arises from the combination of CTD phosphorylation defects and altered regulation of pre‑mRNA processing at intronic polyadenylation sites, making long DDR genes particularly sensitive to reduced CDK12‑dependent elongation and processing. CDK12 also regulates expression of core DNA replication genes and other cell‑cycle regulators by controlling RNAPII processivity over these loci, thereby contributing to proper G1/S progression and coordination between replication and repair. In cancer, recurrent CDK12 loss‑of‑function mutations occur in high‑grade serous ovarian carcinoma, prostate cancer, and other tumor types and associate with tandem duplications, focal genomic rearrangements, and a distinct genomic instability signature that reflects defective transcription of homologous recombination genes rather than direct catalytic lesions in replication or repair enzymes.

References
[1] Krajewska M, et al. Nat Commun. 2019 Apr 15;10(1):1757. [2] Tellier M, et al. Nucleic Acids Res. 2020 Aug 20;48(14):7712-7727.

PVDFメンブレン孔径参考表

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%